Methods and compositions for treatment of peripheral neuropathies

ABSTRACT

A composition for therapy of a peripheral neuropathy disorder in a subject in need thereof. The composition comprises an effective amount of an agent selected from a group consisting of pirenzepine, oxybutynin, muscarinic toxin 7, a muscarinic receptor antagonist, and combinations thereof, and a pharmacologically acceptable carrier and/or an excipient. The composition is useful for therapy of peripheral neuropathies exemplified by peripheral neuropathies induced by systemic diseases, peripheral neuropathies induced by metabolic diseases, chemotherapy-induced peripheral neuropathies, compression-induced peripheral neuropathies, peripheral neuropathies induced by exposure to dichloroacetate, immune-mediated peripheral neuropathies, peripheral neuropathies induced by infections, and genetically acquired peripheral neuropathies.

PRIORITY AND CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 15/106,778, filed Jun. 20, 2016 and published as US 2017/0027957 A1on Feb. 2, 2017, which is a U.S. National Stage Application under 35U.S.C. § 371 of International Application No. PCT/CA2014/051227, filedDec. 17, 2014, designating the U.S. and published in English as WO2015/089664 A1 on Jun. 25, 2015, and which claims the benefit of U.S.Provisional Application No. 61/919,301, filed Dec. 20, 2013. Any and allapplications for which a foreign or a domestic priority is claimedis/are identified in the Application Data Sheet filed herewith andis/are hereby incorporated by reference in their entireties under 37C.F.R. § 1.57.

FIELD OF THE INVENTION

This disclosure relates to compositions for therapeutic treatment ofperipheral neuropathy disorders, to the use of the compositions, and tomethods for use of the compositions.

BACKGROUND

Peripheral neuropathy is a clinical problem in persons affected withdiabetes or treated with chemotherapeutic agents. Peripheralneuropathies can be also caused by infections such as HIV and leprosy.The clinical symptoms may include development of upper back and/orabdominal pain (i.e., thoracoabdominal neuropathy), loss of control ofeye movements (i.e., third-nerve palsy), and progressive loss offunction of the nerves comprising the peripheral nervous system (e.g.,polyneuropathy, mononeuropathy, mononeuritis simplex, autonomicneuropathy). Peripheral neuropathies include the following: neuropathyassociated with diabetes mellitus (diabetic neuropathy), HIV-associatedneuropathy; nutritional deficiency-associated neuropathy; cranial nervepalsies; drug-induced neuropathy; industrial neuropathy; lymphomatousneuropathy; myelomatous neuropathy; multi-focal motor neuropathy;immune-mediated disorders, chronic idiopathic sensory neuropathy;carcinomatous neuropathy; acute pain autonomic neuropathy; alcoholicneuropathy; compressive neuropathy; vasculitic/ischaemic neuropathy;mono- and polyneuropathies. There are a range of genetically inheritedperipheral neuropathies exemplified by Charcot-Marie-Tooth (CMT) diseaseand all its forms, and Friedreich's ataxia. Other peripheralneuropathies may arise from Raynaud's Phenomenon (including CRESTsyndrome), leprosy and autoimmune diseases such as erythromatosis andrheumatoid diseases.

Therapeutic compositions may cause occurrences of peripheralneuropathies, particularly those used for the treatment of neoplasticdisease. In certain cases, peripheral neuropathy is a major complicationof cancer treatment and is the main factor limiting the dosage ofchemotherapeutic agents that can be administered to a patient (Cavalettiet al. 2013, The chemotherapy-induced peripheral neuropathy outcomemeasures standardization study: from consensus to the first validity andreliability findings. Ann. Oncol. 24:454-462). Chemotherapy-inducedperipheral neuropathy (CIPN) often occurs during treatment of variouscancers and other disorders with a variety of agents including taxanes(i.e. paclitaxel/taxol), platinum-based drugs (i.e. cisplatin), vinkaalkaloids (i.e. vincristine) proteasome inhibitors (e.g., Bortezomib)and agents that alter cancer cell metabolism (e.g. dichloroacetate).CIPN can limit dose and duration of treatment, thereby reducing efficacyof the chemotherapeutic regime. Up to 40% of cancer patients treatedwith chemotherapy describe some form of CIPN, with sensory neuropathyfrequently being dominant. Symptoms vary from tingling and numbnessindicative of sensory loss to aspects of painful neuropathy such asallodynia and spontaneous shooting pains that usually start in the handsand feet before moving proximally. Conduction velocity of large sensoryand motor fibers may also be impaired upon electrophysiological testing.CIPN is dose dependent and lowering the dose of the chemotherapeutic orcompletely withdrawing treatment can reduce or eliminate symptoms over aperiod that may be from days to months.

Paclitaxel is a microtubule-stabilizing drug that impedes cell divisionand this is the presumed basis of its chemotherapeutic properties.Paclitaxel is commonly used to treat breast, lung and ovarian cancers,but is dose limited by CIPN. The manifestation of CIPN inpaclitaxel-intoxicated rodents is protocol dependent, possiblyreflecting the dose-related nature of the clinical condition.

Studies of paclitaxel-induced neuropathy in patients and rodents suggestpathogenic mechanisms that include disruption of organelle transport inaxons via microtubule reorganization and damage to Schwann cells andsatellite cells. There is accumulating evidence that paclitaxel, andother instigators of CIPN share a common pathogenic mechanism involvingmitochondrial dysfunction. The resulting energy depletion has thepotential to impede high ATP consuming processes, such as actintreadmilling and consequent peripheral terminal plasticity. Retractionand degeneration of the peripheral terminals of sensory C fibers hasbeen noted in paclitaxel-treated animals.

Dichloroacetate (DCA) is an environmental toxin that is also use totreat mitochondrial diseases by virtue of its ability to inhibitpyruvate dehydrogenase (PDH) kinase, which results in increased PDHactivity and therefore increased flow of pyruvate into the electrontransport chain, with the end result of increasing ATP production. Ithas been recently recognized that DCA can kill certain cancerous cellsby disrupting their inherently anaerobic metabolism. Patients given DCAto treat mitochondrial disease or cancer develop an unwanted side effectof peripheral neuropathy that presents as tingling, loss of sensationand/or pain in the extremities and which can be dose limiting to thepoint of causing cessation of DCA treatment.

The dominant form of neuropathy in the majority of neuropathic diseasesis a distal symmetrical polyneuropathy that initially affects subjects'feet, legs and hands. The primary symptoms include loss of touchingand/or feeling sensations and the loss of ability to sense pain-causingstimuli. A sub-group of patients also develop positive symptoms ofneuropathic pain such as inappropriate tingling, burning, shooting oraching sensations that may co-exist with other negative symptoms ofsensory loss. Such neuropathic pain is commonly referred to as tactileallodynia or mechano-hyperalgesia.

Distal sensory neuropathy is a major component of symmetricalpolyneuropathy and can be measured using skin biopsies to determine lossof intraepidermal nerve fibers (IENF) (Kennedy et al., 1996,Quantitation of epidermal nerves in diabetic neuropathy. Neurology47:1042-48). IENF loss represents retraction of sensory neuron nerveendings from the epidermis with subsequent sensory loss that ultimatelycontributes to high incidences of ulceration, gangrene and amputation insubjects suffering peripheral neuropathy. Currently, there are noregulatory approved therapies available in North America fordegenerative symmetrical polyneuropathy. The current costs to healthsystems for providing relief of these symptoms are enormous.

Muscarinic acetylcholine receptor antagonists block binding ofacetylcholine to muscarinic receptors (G-protein coupled receptors,i.e., GPCRs, with sub types of M1, M2, M3, M4 and M5). Antimuscarinicdrugs were found to treat both the negative (nerve conduction slowingand sensory loss) and positive (allodynia/hyperalgesia) symptoms ofdiabetic symmetrical polyneuropathy, not only ameliorating but alsoreversing nerve damage. Several muscarinic acetylcholine receptorantagonists promote sensory neuron growth in vitro and significantlyprevented and/or reversed loss of intraepidermal and corneal nervefibers, thermal hypoalgesia, large fiber conduction slowing and tactileallodynia, symptoms commonly associated with peripheral neuropathies.Pirenzepine, a selective M1 receptor antagonist that acts as acompetitive inhibitor via interaction with the orthosteric site of thereceptor, proved particularly efficacious. The most specific antagonistof the M1 receptor is muscarinic toxin 7 (MT7), a 64-66 amino acidprotein, derived from the African green mamba snake (Dendroaspisangusticeps). This protein binds to an allosteric site on the M1receptor at low nanomolar concentrations and shows 10,000-foldselectivity for M1 receptors over M2-M5 (Max et al., 1993, Stableallosteric binding of m-toxin to M1 muscarinic receptors. Mol.Pharmacol. 44:1171-5). A less selective compound is the competitiveantagonist oxybutynin (i.e.,4-diethylaminobut-2-ynyl-2-cyclohexyl-2-hydroxy-2-phenyl-ethanoate) iscommonly available in oral compositions and/or transdermal compositionsexemplified by OXYTROL® (OXYTROL is a registered trademark of ActavisInc., Parsippany, N.J., USA), DITROPAN®, DITROPAN XL® (DITROPAN andDITROPAN XL are registered trademarks of Alza Corp., Mountain View,Calif., USA), GELNIQUE® (GELINQUE is a registered trademark of ActavisInc., Parsippany, N.J., USA), Lyrinel XL, Ditrospam, and Urotrol.Oxybutynin is commonly prescribed to relieve urinary and bladderdisorders, including but not limited to urinary incontinence, overactivebladder, enuresis, neuropathic bladder, nephrotuberculosis, neurogenicbladder, and detrusor overactivity. Oxybutynin is also effective fortreating and/or managing postoperative pain related to an indwellingbladder catheter, hyperhidrosis, and refractory hot flashes in cancerpatients. Oxybutynin acts as a selective competitive antagonist ofacetylcholine at muscarinic receptors (M1, M2 and M3), resulting inrelaxation of smooth muscle.

SUMMARY

The exemplary embodiments of the disclosure pertain to compositionsuseful for therapy of peripheral neuropathies. The exemplary therapeuticcompositions may comprise one or more of oxybutynin, pirenzepine,muscarinic toxin 7 (MT7), muscarinic receptor antagonists, and the like.Alternatively, the exemplary therapeutic compositions may comprise oneor more of a salt of oxybutynin, a salt of pirenzepine, a salt of MT7, asalt of a muscarinic receptor antagonist, and the like. Alternatively,the exemplary therapeutic compositions may comprise one or more of aderivative of oxybutynin, a derivative of pirenzepine, a derivative ofMT7, a derivative of a muscarinic receptor antagonist, and the like. Thecompositions are suitable for treating both the negative symptoms ofperipheral neuropathy exemplified by nerve conduction slowing and bysensory loss, and the positive symptoms of peripheral neuropathyexemplified by tactile allodynia and by mechano-hyperalgesia.

Other exemplary methods pertain to methods for manufacturing the topicalcompositions of the present disclosure. Other exemplary methods pertainto methods for manufacturing the oral compositions of the presentdisclosure.

DESCRIPTION OF THE DRAWINGS

The present disclosure will be described in conjunction with referenceto the following drawings in which:

FIG. 1 is a chart showing dose-dependent effects of selective M1receptor antagonists on neurite outgrowth from dissociated sensoryneurons that were derived from adult Sprague Dawley rats;

FIGS. 2(A) and 2(B) are charts showing the effects of pirenzepine (PZ)on nerve conduction velocity (NCV) in streptozotocin (STZ)-induceddiabetic rats;

FIGS. 3(A), 3(B), 3(C), 3(D) show the effects of treatment time withpirenzepine on activation of AMP-activated protein kinase (AMPK) incontrol rats (FIGS. 3(A), 3(B)) and diabetic rats (FIGS. 3(C), 3(D));

FIGS. 4(A)-4(D) are micrographs of GFP fluorescence images of adultsensory neuron cultures derived from STZ-induced diabetic rats showingblockade by dominant negative mutants of AMPK of pirenzepine-inducedneurite outgrowth;

FIGS. 5(A), 5(B) are charts showing the total neurite outgrowth in thecultures shown in FIGS. 4(A)-4(D);

FIG. 6 is a chart showing 1 μM pirenzepine enhanced transcriptionalactivity of peroxisome proliferator-activated receptor-γ coactivator-1α(PGC-1α);

FIGS. 7(A), 7(B) are charts showing that sensory neuron respiration(related to mitochondrial-dependent oxidative phosphorylation), wasaugmented in neuronal cultures derived from (A) M1 receptor knockoutmice (M1R KO), of (B) adult cultures from STZ-induced diabetic ratstreated with 1 μM VU0255035;

FIGS. 8(A), 8(B) are charts showing the coupling efficiency in neuronalcultures derived from M1R KO mice (A) and in STZ-induced diabetic ratstreated with VU0255035 (B), FIGS. 8(C), 8(D) are charts showing therespiratory control ratio in neuronal cultures derived from M1R KO mice(C) and in STZ-induced diabetic rats treated with VU0255035 (D), and8(E), 8(F) are charts showing the spare respiratory capacity in neuronalcultures derived from M1R KO mice (E) and in STZ-induced diabetic ratstreated with VU0255035 (F);

FIG. 9(A) is a micrograph of sensory neurons derived from normal adultrats, while FIG. 9(B) is a micrograph of the effects of muscarinic toxin7 (MT7) on neurite outgrowth in these sensory neurons;

FIG. 10 is a chart showing that MT7 dose-dependently elevated totalneurite outgrowth in sensory neurons derived from normal adult rats;

FIGS. 11(A), 11(B) are charts showing the effects of MT7 (A) andco-treatment with MT7 and compound C (the inhibitor of AMPK), (B) ontranscriptional activity of PGC-la in cultured adult sensory neuronsderived from adult STZ-induced diabetic rats;

FIG. 12 is a chart showing the inhibition of pirenzepine-induced neuriteoutgrowth in cultured adult sensory neurons derived from adult rats bythe Ca²⁺/calmodulin-dependent kinase kinase (CaMKK) inhibitor, STO-609;

FIGS. 13(A), 13(B) show the inhibition of pirenzepine-induced activationof AMPK in cultured adult sensory neurons from normal adult rats by theCaMKK inhibitor, STO-609, while FIGS. 13(C), 13(D) show the inhibitionof MT7-induced activation of AMPK in cultured sensory neurons fromnormal adult rats by the CaMKK inhibitor, STO-609;

FIG. 14 is a chart showing that oxybutynin elevated neurite outgrowth incultured adult sensory neurons;

FIG. 15 is a chart showing the ability of oxybutynin to reverse loss ofpaw thermal sensitivity in a mouse model of type 2 diabetes (control is“C57”; diabetic is “db/db”; oxybutynin treated is “db/db+oxy”);

FIG. 16(A) is a chart showing the effects of topical oxybutynin onintraepidermal nerve fiber (IENF) density in genetically diabetic db/dbmice, while FIG. 16(B) is a chart showing the effects of topicaloxybutynin on nerve fibers within the cornea of diabetic db/db mice;

FIG. 17 is a chart showing effects of systemic oxybutynin on developmentof thermal hypoalgesia in STZ-induced diabetic Swiss Webster mice;

FIG. 18(A) is a chart showing that pirenzepine enhanced neuriteoutgrowth in the presence of the chemotherapy agent paclitaxel incultures of adult sensory neurons, while FIG. 18(B) is a chart showingthat pirenzepine enhanced neurite outgrowth in the presence of thechemotherapy agent oxaliplatin in cultures of adult sensory neurons;

FIG. 19 is a chart showing paw thermal response latency (left panel) and50% tactile response threshold (right panel) in mice treated withpaclitaxel (taxol)±pirenzepine (Pz);

FIGS. 20(A), 20(B), 20(C) are charts showing the effects of pirenzepine(Pz) on paw thermal hypoalgesia (A), depletion of paw skin IENF (B) andon onset of MNCV slowing in mice treated with dichloroacetate (DCA)(C);

FIG. 21 is a chart showing the effects of atroprine delivered topicallyto the hind paws or eyes of STZ-induced diabetic mice on MNCV;

FIG. 22 is a chart showing the effects of atroprine delivered topicallyto the hind paw or eyes of STZ-induced diabetic mice on paw thermallatency;

FIG. 23 is a chart showing the effects of 11 days of topical applicationof MT7 to the eye of control mice on nerve fiber occupancy in thesub-basal nerve plexus (SBNP 1-5) and stroma (Stroma 1-10) of thecornea; and

FIG. 24 is a chart showing protective effect of pirenzepine and MT7 onneurite outgrowth in an in vitro model of HIV-induced neuropathy.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the meanings that would be commonly understood by one of skill inthe art in the context of the present specification. Although anymethods and materials similar or equivalent to those described hereincan also be used in the practice or testing of the present disclosure,the preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural references unless thecontext clearly dictates otherwise. Thus, for example, reference to “anagent” includes a plurality of such agents and reference to “thesubject” includes reference to one or more subjects and equivalentsthereof known to those skilled in the art, and so forth.

“Optional” or “optionally” or “alternatively” means that thesubsequently described event, circumstance, or material may or may notoccur or be present, and that the description includes instances wherethe event, circumstance, or material occurs or is present and instanceswhere it does not occur or is not present.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also, encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

“Inhibit”, “inhibiting”, and “inhibition” mean to decrease an activity,response, condition, disease, or other biological parameter. This caninclude but is not limited to the complete ablation of the activity,response, condition, or disease. This may also include, for example, a10% reduction in the activity, response, condition, or disease ascompared to the native or control level. Thus, the reduction can be a10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%; 100%, or any amount ofreduction in between the specifically recited percentages, as comparedto native or control levels.

“Promote”, “promotion”, and “promoting” refer to an increase in anactivity, response, condition, disease, or other biological parameter.This can include but is not limited to the initiation of the activity,response, condition, or disease. This may also include, for example, a10% increase in the activity, response, condition, or disease ascompared to the native or control level. Thus, the increase in anactivity, response, condition, disease, or other biological parametercan be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more,including any amount of increase in between the specifically recitedpercentages, as compared to native or control levels.

As used herein, the term “subject” means any target of administration.The subject can be a vertebrate, for example, a mammal. Thus, thesubject can be a human. The term does not denote a particular age orsex. Thus, adult, juvenile, and newborn subjects, whether male orfemale, are intended to be covered. A patient refers to a subjectafflicted with a disease or disorder. The term “patient” includes humanand veterinary subjects.

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

The term “therapeutically effective” means that the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the subjectto be treated.

As used herein, “pharmaceutical composition” includes any compositionfor: (i) topical administration, or (ii) transdermal administration or(iii) parenteral administration, or (iv) oral administration, ofoxybutynin, pirenzepine, muscarinic toxin 7 (referred to herein after as“MT7”), muscarinic receptor antagonist, and the like to a subject inneed of therapy for peripheral neuropathy. Pharmaceutical compositionsmay include carriers, thickeners, diluents, buffers, preservatives,surface active agents and the like in addition to oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like.Pharmaceutical compositions may also include one or more activeingredients such as antimicrobial agents, anti-inflammatory agents,anaesthetics, analgesics, and the like.

Muscarinic acetylcholine receptor antagonists reverse loss ofintraepidermal nerve fibers and thermal hypoalgesia in peripheralneuropathies. Muscarinic acetylcholine receptor antagonists are agentsthat reduce the activities and/or function of muscarinic acetylcholinereceptors that are found in the plasma membranes of neurons and othercells. Muscarinic acetylcholine receptors are GPCRs that are stimulatedby acetylcholine released from several cell types including sensoryneurons, keratinocytes and postganglionic fibers in the parasympatheticnervous system, and function as signaling molecules that initiate signalcascades within cells in their immediate regions. Well-known muscarinicacetylcholine receptor antagonists useful for treatment of maladies suchas central nervous system malfunctioning, pulmonary diseases, andgastric ailments are exemplified by atropine, scopolamine, pirenzepine,telenzepine, hyoscine, hyoscyamine, ipratropium, tropicamide,cyclopentolate, glycopyrrolate,4-diphenylacetoxy-1,1-dimethylpiperidinium, quinidine, orphenadrine,oxybutynin, oxyphenonium, emepronium, procyclidine, propantheline,4-fluorhexahydrosiladifenidol, octylonium, quinuclidinyl benzilate,tolterodine, benactyzine, fesoterodine (fumarate), trospium,solifenacin, gallamine, bipreiden, dicyclomine, benztropine, dexetimide,hexahydrosiladifenidol, among others.

Furthermore, in addition to pirenzepine, there are a number of selectiveantagonists for the type 1 muscarinic receptor (M1R) and othermuscarinic receptor subtypes that are anticipated to exhibit beneficialactivities similar to those demonstrated by pirenzepine on neuronalneurite outgrowth. Some of these compounds have much superior MIRselective activity. For example, telenzepine, an analog of pirenzepinewith an altered tricyclic structure but an unmodified piperazine sidechain, is 4 to 10 times more potent than pirenzepine. VU0255035, athiadiazole derivative and is 75 times more selective to MIR relative toM2, M3, M4 and M5 receptors. Among the new generation of MIRantagonists, promising centrally active MIR antagonists includePD150714, and 77-LH-28-1 and spirotramine. Listings of muscarinicacetylcholine receptor antagonists suitable for incorporation intotherapeutic compositions for peripheral neuropathy include: muscarinictoxin 7 (MT7) green mamba venom; tricyclic benzodiazepinone derivatives(such as pirenzepine, telenzepine); 1,4-disubstituted tetrahydropyridinecarboxylic acids exemplified by PD150714; trihexyphenidyl analogsexemplified by trihexyphenidyl and p-fluorotrihexyphenidy; thiadiazolesulfonamide derivatives exemplified by VU0255035; hexocyclium andsila-hexocyclium exemplified by 0-methoxy-sila-hexocyclium;polymethylene tetraamine orspiro-4-damp(4-diphenyl-acetoxy-n-methylpiperidine exemplified byspirotramine; n-(4-(4-ethylpiperazin-1-yl) phenyl amide analogues;McN-A-343 analogues; alkoxy-oxadiazolyltetrahydropyridines exemplifiedby MB-OXTP; caramiphen, aprophen and related derivatives exemplified bynitrocaramiphen and aprophen; (−)-S-ET126; N-desmethylclozapine;MDL74019DG; glycopyrronium bromide; and dicyclomine. Also included areMIR mixed antagonists, i.e. compounds that show antagonist effects atmore than one subtype of muscarinic receptor, including M1, such asrispenzepine, R-procyclidine, and DAU 5750. Other muscarinic antagonistsinclude nuvenzepine, 4-fluorohexahydrosiladifenidol,4-diphenylacetoxy-N-methyl-piperidine methiodide, tolterodine, PD102807,oxybutynin, iptratropium bromide, and the like.

Variations to the structures of the above descriptions of muscarinicantagonists could make these compounds more selective MIR antagonists,or alternatively, be varied for stability, safety or efficacy.Accordingly, generic formulae can be designed that encompass thestructural features for the antagonists, including those that are MIRselective and M1R-non-selective compounds. Suitable formulae areexemplified by Formula I and Formula II.

R1 may be one of a 5-membered unsaturated ring, a 6-membered unsaturatedring, or a hetero atom-containing ring;

R2 may be one of a 5-membered unsaturated ring, a 6-membered unsaturatedring, or a hetero atom-containing ring;

R3 may be one of a H-piperidinyl group, a 2-piperidinyl group, a3-piperidinyl, a 4-piperidinyl group, a 2-piperazinyl group, or a3-piperazinyl group, linked via a methyl group or an ethyl group or apropyl group or a butyl group. The piperidinyl groups or piperazinylgroups may additionally be linked to methyl, trifluoromethyl or ethylmoieties.

R4 may be a hydrogen ion or a chloride ion.

R5 may be a hydrogen ion or a chloride ion.

R6 may be a hydrogen ion or a chloride ion.

R7 may be a hydrogen ion or a chloride ion.

X may be a methyl group or a “NR” group i.e. a primary amine group, asecondary amine group, or a tertiary amine group.

R1 may be

wherein “ ” indicates the point of connection of R1 with the upperstructure in Formula II.

R2 may be a hydroxyl ion or a hydrogen ion or a ketone.

R3 may be a hydroxyl ion or a hydrogen ion or a ketone.

The term “unit dosage form” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the likecalculated in an amount sufficient to produce the desired effect inassociation with a pharmaceutically acceptable diluent, carrier orvehicle.

The term “carrier” means a compound, composition, substance, orstructure that, when in combination with oxybutynin, pirenzepine, MT7,muscarinic receptor antagonist, and the like or composition, aids orfacilitates preparation, storage, administration, delivery,effectiveness, selectivity, or any other feature of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like orcomposition for its intended use or purpose. For example, a carrier canbe selected to minimize any degradation of the oxybutynin, pirenzepine,MT7, muscarinic receptor antagonist, and the like and to minimize anyadverse side effects in the subject.

Suitable pharmaceutically acceptable carriers include essentiallychemically inert and nontoxic pharmaceutical compositions that do notinterfere with the effectiveness and/or safety of the primary biologicalactivity of the pharmaceutical composition. Suitable carriers and theirformulations are described in Remington (1995, The Science and Practiceof Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company,Easton, Pa.). Typically, an appropriate amount of a pharmaceuticallyacceptable salt is used in the formulation to render the formulationisotonic. Examples of suitable pharmaceutical carriers include, but arenot limited to, saline solutions, glycerol solutions, ethanol,N-(1(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),diolesylphosphotidylethanolamine (DOPE), and liposomes. Suchpharmaceutical compositions should contain a therapeutically effectiveamount of the compound, together with a suitable amount of carrier so asto provide the form for proper administration to the subject. Theformulation should suit the mode of administration. For example, oraladministration requires enteric coatings to protect oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like fromdegradation within the gastrointestinal tract. In another example, theoxybutynin, pirenzepine, MT7, muscarinic receptor antagonist, and thelike may be administered in a liposomal formulation to facilitatetransport throughout a subject's vascular system and effect deliveryacross cell membranes to intracellular sites.

The term “excipient” herein means any substance, not itself atherapeutic agent, which may be used in a composition for delivery ofoxybutynin, pirenzepine, MT7, muscarinic receptor antagonist, and thelike to a subject or alternatively combined with oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like (e.g., tocreate a pharmaceutical composition) to improve its handling or storageproperties or to permit or facilitate formation of a dose unit of thecomposition (e.g., formation of a topical hydrogel which may then beoptionally incorporated into a transdermal patch). Excipients include,by way of illustration and not limitation, binders, disintegrants, tasteenhancers, solvents, thickening or gelling agents (and any neutralizingagents, if necessary), penetration enhancers, solubilizing agents,wetting agents, antioxidants, lubricants, emollients, substances addedto mask or counteract a disagreeable odor, fragrances or taste,substances added to improve appearance or texture of the composition andsubstances used to form the pharmaceutical compositions. Any suchexcipients can be used in any dosage forms according to the presentdisclosure. The foregoing classes of excipients are not meant to beexhaustive but merely illustrative as a person of ordinary skill in theart would recognize that additional types and combinations of excipientscould be used to achieve the desired goals for delivery of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like.

Peripheral neuropathy is an urgent, unmet clinical need. Nervedegeneration and impaired regeneration are pathological hallmarks ofperipheral neuropathy. Diabetic neuropathy most frequently manifests asa distal symmetrical polyneuropathy, afflicting both the somatic andautonomic divisions of the peripheral nervous system. Diabeticneuropathy is often initially described as a small fiber neuropathy andskin biopsies reveal the early retraction of small sensory fibers fromthe epidermis (Said, 2007, Diabetic neuropathy—a review. Nat. Clin.Pract. Neurol. 3:331-340). Segmental demyelination and axonaldegeneration of large fibers are seen in nerve biopsies from patientsafter many years of diabetes, along with clusters of regenerating smallfibers (Kalichman et al., 1998, Reactive, degenerative, andproliferative Schwann cell responses in experimental galactose and humandiabetic neuropathy. Acta Neuropath. 95:47-56). The capacity of injuredsmall fibers to regenerate has been shown to be impaired in repeatedskin biopsies from diabetic subjects (Polydefkis et al. 2004, The timecourse of epidermal nerve fibre regeneration: studies in normal controlsand in people with diabetes, with and without neuropathy. Brain127:1606-1615). It appears that peripheral nerves are in a constantdynamic equilibrium between nerve retraction and re-growth and that thediverse metabolic stresses of systemic diabetes enhance retraction andimpede regeneration, leading to distal nerve degeneration.

Peripheral neuropathy is generally recognized by patients at onset ofsensory symptoms such as pain, dysesthesia's and/or sensory loss in theextremities. Physicians can confirm the diagnosis using simple sensorytests (15 g monofilament and tuning fork) or comprehensive scoringsystems that encompass pain scores, sensory and autonomic functiontests, and detailed large fiber electrophysiology. Historically, mostclinical trials of therapies designed to prevent or alleviate peripheralneuropathy have used large fiber electrophysiology as the primary endpoint for efficacy, even where therapies have targeted small fiberneuropathy. Trials have occasionally included sural nerve biopsies toevaluate the pathology underlying symptoms but this approach is invasiveand not recommended. There is an emerging focus on small fiber pathologyto measure peripheral neuropathy and efficacy of interventions, as smallsensory fiber terminals can be assessed quantitatively and iterativelyin longitudinal studies using minimally invasive skin biopsy ornon-invasive corneal confocal microscopy techniques. IENF and cornealnerve depletion, representing mostly small unmyelinated sensory fibersthat transduce thermal sensation, correlates well with symptoms such aspain and sensory loss and also with measures of large fiber neuropathysuch as conduction slowing. Moreover, rodent models of peripheralneuropathy also develop IENF and corneal nerve depletion. This offers alogical and scientifically consistent pathway for development oftherapies that promote nerve regeneration after injury, starting with invitro studies to assess potential for promoting axonal growth after thetrauma of dissecting dorsal root ganglia (DRG), then in vivo studies inrodent models of peripheral neuropathy to assess IENF numbers andfunction and then clinical studies that measure the same parameter.

The term “neuropathy” includes any pathology or abnormality of neuraltissue causing nerve dysfunction. The function of the nerve or nervesthat is disrupted may involve the rate of flow of the electrical currentthrough the nerve or may involve ectopic firing (firing in the absenceof stimulus) of the nerve, or may involve inappropriate or inadequatefiring of the nerve in response to a stimulus. Peripheral neuropathy asused herein is defined as a disorder resulting from damage to peripheralnerves. It may be acquired, caused by diseases of the nerves or as theresult of systemic illness.

A number of factors can cause, induce or are associated with peripheralneuropathies, and included within the scope of the disclosure areperipheral neuropathies associated with, diabetes, infectionsexemplified by HIV, toxic agents, alcoholism, nutritional deficiencies,systemic/metabolic disorders, palsies, auto-immune disorders, inheritedor genetic disorders, cancers and tumors, compressive neuropathies;vasculitic/ischaemic neuropathy; mono- and polyneuropathies. In furtherembodiments, the peripheral neuropathy manifests as a post surgicalcomplication.

Preferably included within the scope of the term neuropathy/neuropathiesare neuropathies associated with diseases such as: uremia; childhoodcholestatic liver disease; chronic respiratory insufficiency; alcoholicpolyneuropathy; multiple organ failure; sepsis; hypoalbuminemia;eosinophilia-myalgia syndrome; hypoglycemia; vitamin or nutritionaldeficiency (e.g., B-12 deficiency, vitamin A deficiency, vitamin Edeficiency, vitamin B1 deficiency); primary biliary cirrhosis;hyperlipidemia; sensory perineuritis; allergic granulomatous angiitis;hypersensitivity angiitis; Bell's Palsy, Wegener's granulomatosis;rheumatoid arthritis; systemic lupus erythematosis; mixed connectivetissue disease; scleroderma; systemic vasculitides; acute tunnelsyndrome; pandysautonomia; hypothyroidism; chronic obstructive pulmonarydisease; acromegaly; malabsorption (sprue, celiac disease); carcinomas(sensory, sensorimotor, late and demyelinating); lymphoma (includingHodgkin's), polycythemia vera; multiple myeloma (lytic type,osteosclerotic, or solitary plasmacytoma); tropical myeloneuropathies;pernicious anemia, Churg-Strauss syndrome; cranial nerve palsies;drug-induced neuropathy; industrial neuropathy; lymphomatous neuropathy;myelomatous neuropathy, chronic idiopathic sensory neuropathy;carcinomatous neuropathy; acute pain autonomic neuropathy; compressiveneuropathy; mono- and polyneuropathies; or diabetes.

In preferred embodiments, the peripheral neuropathy is a diabeticneuropathy. It will be clearly understood that the diabetic neuropathymay be diabetic or pre-diabetic associated with Type 1(insulin-dependent) diabetes, Type 2 (non-insulin-dependent) diabetes,or both.

In other embodiments the peripheral neuropathy is induced by oralternatively, a secondary affect due to a toxic agent such as a drug,industrial chemical or environmental toxin. For example, the peripheralneuropathy can be caused by a chemotherapeutic agent such as paclitaxel(or other taxane derivative), alkaloids such as vincristine orvinblastin, platinum compounds such as cisplatin, carboplatin,oxaliplatin, dichloroacetate, topoisomerase inhibitors, intercalatorssuch as bleomycin, or drugs such as chloramphenicol, colchicine,dapsone, disulfiram, amiodarone, gold, isoniazid, misonidazole,nitrofurantoin, perhexiline, propafenone, pyridoxine, phenytoin,simvastatin, tacrolimus, thalidomide, cyclophosphamide or zalcitabine,an agent used for the treatment of infectious diseases such asstreptomycin, didanosine or zalcitabine, or any other chemically toxicagent such as acrylamide, arsenic, carbon disulfide, hexacarbons, lead,mercury, platinum, an organophosphate, thallium, or alcohol.

In another preferred embodiment, the peripheral neuropathy caused by asystemic or metabolic disease is selected from the group consisting ofdiabetic or prediabetic neuropathy, acquired primary demyelinatingneuropathy, distal symmetric sensory polyneuropathy, distal symmetricsensorimotor polyneuropathy, vasculitic neuropathy, infectiousneuropathy, idiopathic neuropathy, immune-mediated neuropathy,nutrition-related neuropathy, kidney or liver failure, andparaneoplastic neuropathy.

In other embodiments, the peripheral neuropathy is induced by aninfection or infectious disease, such as leprosy, Lyme disease, HIV oracquired immunodeficiency syndrome (AIDS), post-polio syndrome, herpessimplex and herpes zoster (aka shingles); hepatitis B, hepatitis C, HIV,cytomegalovirus, or diphtheria.

In a preferred embodiment, immune-mediated such as acquired primarydemyelinating neuropathy includes chronic inflammatory demyelinatingpolyradiculoneuropathy (CIDP), Guillain-Barre syndrome/acuteinflammatory demyelinating polyneuropathy (AIDP), sarcoidosis;vasculitic/ischaemic neuropathy (such as polyarteritis nodosa,rheumatoid arthritis, systemic lupus erythematosus (Lupus) and Sjogren'ssyndrome, celiac disease (sprue), multi-focal motor neuropathy (MNN), orperipheral neuropathy associated with protein abnormalities (such asmonoclonal gammopathy, amyloidosis, cryoglobulinemia, macroglobulinemia,POEMS).

In a preferred embodiment, the compression that causes peripheralneuropathy is selected from the group consisting of carpal tunnelsyndrome, ulnar neuropathy at the elbow or wrist, common peroneal nerveat the knee, tibial nerve at the knee, amyloidosis, and sciatic nerve.

Also included within the scope of the term neuropathy/neuropathies arehereditary or genetically acquired neuropathies, including peronealmuscular atrophy (Charcot-Marie-Tooth Disease) hereditary amyloidneuropathies, hereditary sensory neuropathy (type I and type II),porphylias or porphyric neuropathy, hereditary (neuropathy) liability topressure palsy (HNPP), Fabry's Disease, adrenomyeloneuropathy, Riley-DaySyndrome, Dejerine-Sottas neuropathy (hereditary motor-sensoryneuropathy-III), Refsum's disease, Raynaud's disease including CRESTsyndrome. Krabbe's disease, ataxia-telangiectasia, hereditarytyrosinemia, anaphalipoproteinemia, abetalipoproteinemia, giant axonalneuropathy, metachromatic leukodystrophy, globoid cell leukodystrophy,or Friedrich's ataxia.

The compositions and methods of the disclosure can be also be used totreat or prevent neuropathy related to or induced by the followingdiseases, trauma, or conditions: general neuropathic conditions, such asperipheral neuropathy, phantom limb pain, reflex-sympathetic dystrophy,causalgia, syringomyelia, and painful scar; specific neuralgias at anylocation of the body; back pain; diabetic neuropathy; alcoholicneuropathy; metabolic neuropathy; inflammatory neuropathy;chemotherapy-induced neuropathy, herpetic neuralgias; traumaticodontalgia; endodontic odontalgia; thoracic-outlet syndrome; cervical,thoracic, or lumbar radiculopathies with nerve compression; cancer withnerve invasion; traumatic-avulsion injuries; mastectomy, thoracotomypain; spinal-cord-injury; stroke; abdominal-cutaneous nerve entrapments;tumors of neural tissues; arachnoiditis; stump pain; fibromyalgia;regional sprains or strains; myofascial pain; psoriatic arthropathy;polyarteritis nodosa; osteomyelitis; burns involving nerve damage;AIDS-related pain syndromes; connective tissue disorders, such assystemic lupus erythematosis, systemic sclerosis, polymyositis, anddermatomyositis; and inflammatory conditions, such as acute inflammation(e.g. trauma, surgery and infection) or chronic inflammation (e.g.,arthritis and gout).

Peripheral nerves undergo continuous cholinergic constraint.Structurally distinct and selective (e.g. pirenzepine) or specific (e.g.MT7) M1 muscarinic receptor antagonists promote neurite outgrowth inprimary cultures of sensory neurons derived from adult rodents.Importantly, neurite outgrowth is also enhanced in cultured sensoryneurons derived from mice lacking the M1 receptor compared to thosederived from normal mice. Along with preclinical data shown below, thisunderlies the general hypothesis that peripheral sensory neurons existunder “cholinergic constraint” that prevents excessive growth ofperipheral terminals. This cholinergic constraint may use autocrineand/or paracrine secretion mechanisms involving the neurotransmitteracetylcholine (the primary endogenous ligand for muscarinic receptors).Adult rat sensory neurons contain a peripheral form of the acetylcholine(ACh) synthesizing enzyme choline acetyltransferase (pChAT), exhibitChAT activity and express both the vesicular ACh transporter and the M1receptor. This explains why M1 receptor antagonists show efficacy in thecell culture systems. A paracrine mechanism may also operate asepidermal keratinocytes and cells of the cornea secrete ACh andcommunicate via cholinergic receptors.

This concept of endogenous restraint of axon growth has some precedencein studies of the CNS. Moreover, preclinical data in type 1 and type 2diabetic rodents and rodents with CIPN and DCA-induced neuropathydemonstrates that a therapeutic strategy in which neurons are protectedfrom degeneration and encouraged towards regeneration by targeting theendogenous cholinergic constraint system using muscarinic receptorantagonists is viable. Muscarinic receptor antagonists are not new drugsand a number are in clinical use throughout the world to treat a varietyof diseases separate and distinct from diabetic and other peripheralneuropathies.

A common feature that unifies the etiology of nerve degeneration innumerous peripheral nerve diseases involves impaired mitochondrialfunction. Inability of mitochondria to produce sufficient ATP leads tonerve degeneration and the failure of nerves to regenerate after stressand/or damage (Roy Chowdhury et al., 2013, The role of aberrantmitochondrial bioenergetics in diabetic neuropathy. Neurobiol. Dis. 51:56-65).

Diabetic Neuropathy:

Sensory neurons in animal models of type 1 and type 2 diabetes exhibitimpaired mitochondrial gene expression and function. Studies on neuronsderived from diabetic rodents reveals that their mitochondrial membranepotentials are depressed (Huang et al., 2003, Insulin preventsdepolarization of the mitochondrial inner membrane in sensory neurons oftype 1 diabetic rats in the presence of sustained hyperglycemia.Diabetes 52:2129-36). Analysis of the bioenergetics of sensory neuronsfrom diabetic rodents shows sub-optimal maximal respiration capacity andloss of activity of electron transport complexes (Roy Chowdhury et al.,2012, Impaired AMP-activated protein kinase signaling in dorsal rootganglia neurons is linked to mitochondrial dysfunction and peripheralneuropathy in diabetes. Brain 135:1751-66). Changes in the mitochondrialproteome underlie these alterations and are driven by impairedactivation of AMP-activated protein kinase (AMPK)—a master regulator ofmitochondrial function and fidelity (Roy Chowdhury et al., 2012; Akudeet al., 2011, Diminished superoxide generation is associated withrespiratory chain dysfunction and changes in the mitochondrial proteomeof sensory neurons from diabetic rats. Diabetes 60:288-97). In diabeticneuropathy, this down-regulation of mitochondrial function in adultsensory neurons is associated with suppression of AMPK and PGC-laactivity (Roy Chowdhury et al. 2012, Impaired adenosinemonophosphate-activated protein kinase signaling in dorsal root ganglianeurons is linked to mitochondrial dysfunction and peripheral neuropathyin diabetes. Brain 135: 1751-66). The mechanism of M1 receptor mediatedinhibition of axonal growth involves the down-regulation ofmitochondrial bioenergetics and function. Releasing neurons from thisconstraint using M1 receptor antagonists leads to activation of AMPK,increased transcriptional activity of PGC-la and associated enhancementof mitochondrial respiration. The M1 receptor modulates this pathway ata proximal level via CaMKKO (or CaMKK2), a described activator of AMPK.AMPK is a multi-component Ser/Thr kinase activated by binding of AMPupon a rise in the AMP/ATP ratio. Activated AMPK switches on catabolicpathways, primarily through optimization of mitochondrial function, toproduce ATP while simultaneously shutting down energy-consuming anabolicprocesses. AMPK activation increases phosphorylation of thetranscription factor PGC-la and AMPK requires PGC-la activity tomodulate the expression of several key players in metabolism, includingcomponents of the mitochondrial electron transport system. Deacetylationof PGC-1α by the cytoplasmic sirtuins, SIRT1 and SIRT2, increases itstranscriptional activity. Coupled regulation of PGC-lu by AMPK andsirtuins plays a major role in the metabolic adaptations to energymetabolism in different tissues.

The positive effects of pirenzepine on neurite outgrowth,phosphorylation of AMPK and transcriptional activation of PGC-la arereplicated by MT7. Oxybutynin also elevates neurite outgrowth incultured sensory neurons and, like pirenzepine, protects mice with type1 and type 2 diabetes from development of sensory neuropathy.

Chemotherapy-Induced Peripheral Neuropathy (CIPN):

Paclitaxel and oxaliplatin induce CIPN through disruption ofmitochondrial function that drives distal axonal loss. (Bennett et al.,2011, Terminal arbor degeneration—a novel lesion produced by theantineoplastic agent paclitaxel. Eur. J. Neurosci. 33:1667-76; Xiao etal., 2011, Mitochondrial abnormality in sensory, but not motor, axons inpaclitaxel-evoked painful peripheral neuropathy in the rat. Neuroscience199:461-9; Zheng et al., 2011, Functional deficits in peripheral nervemitochondria in rats with paclitaxel-and oxaliplatin-evoked painfulperipheral neuropathy. Exp. Neurol. 232:154-61). In rodent models ofCIPN analysis of mitochondrial electron transport activity revealeddeficits in capacity and was linked to dying back of axons in the skin(Zheng et al., 2011). Pirenzepine protected cultured neurons fromdegeneration induced by the CIPN-inducing agents paclitaxel andoxaliplatin. In vivo studies with mice treated with paclitaxel revealedpirenzepine protected from development of thermal and tactile allodynia.Pirenzepine also protected DCA-treated mice from development of thermalhypoalgesia, IENF loss and MNCV slowing.

Charcot-Marie-Tooth (CMT):

In Charcot-Marie-Tooth disease type 2 (CMT2) a distal dying-back axonaldegeneration is predominant and in 19% to 33% of cases, has been linkedto mutations in the GTPase and mitochondrial fusion protein, mitofusin 2(MFN2) (Zuchner, et al., 2004, Mutations in the mitochondrial GTPasemitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nat. Genet.36:449-51). Mutant MFN2 over-expressed in sensory neurons also resultedin a distal dying-back neuropathy in mouse models that was characterizedby compromised axonal trafficking of mitochondria (Baloh et al., 2007,Altered axonal mitochondrial transport in the pathogenesis ofCharcot-Marie-Tooth disease from mitofusin 2 mutations. J. Neurosci.27:422-30). The bioenergetics properties of mitochondria were notsignificantly altered and the role of MFN2 in mediating transport wasdeemed to be discrete from regulation of mitochondrial fusion (Baloh etal., 2007; Misko et al., 2010, Mitofusin 2 is necessary for transport ofaxonal mitochondria and interacts with the Miro/Milton complex. J.Neurosci. 30:4232-40).

HIV-Induced Neuropathy

Post-mortem peripheral nerve samples from patients with HIV-inducedneuropathy reveal increased mutation in mitochondrial DNA (mutationmtDNA⁴⁹⁷⁷) that was associated with deficits in mitochondrial proteinexpression (Lehmann et al., 2011, Mitochondrial dysfunction in distalaxons contributes to human immunodeficiency virus sensory neuropathy.Ann Neurol. 69, 100-10). These insufficiencies were more pronounced at adistal level compared with more proximal nerve segments. Simianimmunodeficiency virus (SIV) infected macaques exhibited similarabnormalities, with abnormal markers of mitochondrial function and anelevation in mitochondrial-dependent reactive oxygen species (ROS)production (Lehmann et al., 2011). Studies with cultured human DRGtreated with supernatants from HIV-infected macrophages also revealmitochondrial dysfunction (manifesting as membrane depolarization) withsigns of oxidative stress in perikarya but not in axons (Hahn et al.,2008, Differential effects of HIV infected macrophages on dorsal rootganglia neurons and axons. Exp. Neurol. 210:30-40). The alterations inmitochondrial phenotype (e.g., inner membrane depolarization anddepressed expression of proteins), mirror the defects seen in diabeticneuropathy.

Friedreich Ataxia:

This is an autosomal recessive neurodegenerative disease induced by aGAA repeat expansion in intron 1 of the frataxin gene. The resultingdiminished expression is linked with a dying-back neuropathy impactingsensory neurons and spinocerebellar and corticospinal motor tracts(Puccio et al., 2002, Friedreich ataxia: a paradigm for mitochondrialdiseases. Curr. Opin. Genet. Dev. 12:272-77). Frataxin is an ironchaperone required for formation of iron-sulfur (Fe—S) clusters, but itsloss is associated with both diminished activity of Fe—S-containingenzymes (important for optimal mitochondrial respiratory chain function)and with deficient defenses against oxidative stress (Bencze et al.,2006, The structure and function offrataxin. Crit. Rev. Biochem. Mol.Biol. 41:269-91). In mouse models of the disease, mitochondrialdysfunction occurs in the absence of any enhancement of oxidative stress(Seznec et al., 2005, Friedreich ataxia: the oxidative stress paradox.Hum. Mol. Genet. 14:463-74). Some clarity on the etiology of thisdisease has come from work in Drosophila where mitochondrial innermembrane depolarization preceded impaired mitochondrial trafficking anddistal loss of fibers in the absence of oxidate stress (Shidara &Hollenbeck, 2010, Defects in mitochondrial axonal transport and membranepotential without increased reactive oxygen species production in aDrosophila model of Friedreich ataxia. J. Neurosci. 30:11369-78). Thereare distinct parallels with mitochondrial dysfunction observed indiabetic neuropathy. Inner membrane depolarization in sensory neurons isidentified early in the disease in animal models and respiratory chaindysfunction is observed in the absence of any attendant elevation in ROSproduction (Akude et al., 2011; Huang et al, 2003; Roy Chowdhury et al,2012).

Antimuscarinic drugs can protect peripheral nerves from degeneration ina number of common diseases by manipulating cellular AMPK levels andtherefore mitochondrial function. Data is presented below showing thatthese drugs can repair nerve damage in diabetes, CIPN, HIV and DCAtoxicity. These are diseases caused by very different initial stressorsthat share a common mitochondrial component in the etiology orprogression of disease. Importantly, the ability of these drugs torepair mitochondrial fidelity and drive nerve regeneration occursindependent from any role for muscarinic signaling in the etiology ofthe disease. The inhibition of the cholinergic constraint pathway bythese drugs enables nerve regeneration and repair to be enhanced acrossa broad range of diseases.

This disclosure provides compositions comprising agents that include,but are not limited to, oxybutynin, pirenzepine, MT7, muscarinicreceptor antagonist, and the like to treat peripheral neuropathy inducedby diabetes, chemotherapeutic agents including DCA, HIV, and geneticdiseases exemplified by Charcot-Marie-Tooth disease. The agents can beformulated as pharmaceutical compositions for various routes ofdelivery.

In another embodiment, agents exemplified by oxybutynin, pirenzepine,MT7, other muscarinic receptor antagonists and the like are used torestore reduced AMPK activity and PGC-la transcriptional activity.

Each of the foregoing agents (e.g., oxybutynin, pirenzepine, MT7,muscarinic receptor antagonist, and the like), can be formulated in apharmaceutical composition for various routes of administration.

In one embodiment, the pharmaceutical compositions disclosed hereincomprise an agent as described above (e.g., oxybutynin, pirenzepine,MT7, muscarinic receptor antagonist, and the like), in a total amount byweight of the composition of about 0.1% to about 95%. For example, theamount of oxybutynin, pirenzepine, MT7, muscarinic receptor antagonist,and the like, by weight of the pharmaceutical composition may be about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about1.9%, about 2%, about 2.1%>, about 2.2%, about 2.3%, about 2.4%, about2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about3.7%, about 3.8%, about 3.9%, about 4%, about 4.1%, about 4.2%, about4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6%, about6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about6.7%, about 6.8%, about 6.9%, about 7%, about 7.1%, about 7.2%, about7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about7.9%, about 8%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9%, about9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about9.7%, about 9.8%, about 9.9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90% or about 95%.

The pharmaceutical compositions of the disclosure comprising agent(s)may be formulated for topical administration or alternatively, fortransdermal administration.

A pharmaceutical composition for topical administration may be providedas, for example, ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, hydrogels, sprays, aerosols, dressings, oroils. When formulated in an ointment, the active ingredient may beemployed with either a paraffmic or a water-miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water base or a water-in-oil base. Other formulations thecompositions can be incorporated into include oils, suppositories,foams, liniments, aerosols, buccals, and sublingual tablets or topicaldevices for absorption through the skin or mucous membranes.

Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents. Liquid sprays are conveniently delivered frompressurized packs, for example, via a specially shaped closure.Oil-In-Water emulsions can also be utilized in the compositions,patches, bandages and articles. These systems are semisolid emulsions,micro-emulsions, or foam emulsion systems. Usually such a system has a“creamy white” appearance. The oleaginous phase may contain, but is notlimited to, long-chain alcohols (cetyl, stearyl), long-chain esters(myristates, palmitates, stearates), long-chain acids (palmitic,stearic), vegetable and animal oils and assorted waxes. These can bemade with anionic, cationic, nonionic or amphoteric surfactants, or withcombinations especially of the nonionic surfactants. A typical inventiongel base, provided herein for exemplary purposes only, can containlecithin, isopropyl palmitate, poloxamer 407, and water. Topicalcarriers with different viscosities and hand-feel are known to the art.The above active ingredients can be dispersed within thepharmaceutically acceptable carrier in therapeutically effective amountsto treat neuropathies, and the other maladies described above.

A pharmaceutical composition for transdermal administration may beprovided as, for example, a hydrogel comprising agents as describedherein incorporated into an adhesive patch composition intended toremain in intimate contact with a subject's epidermis for a prolongedperiod of time. An exemplary adhesive patch composition can comprise amonolithic layer produced by mixing oxybutynin, pirenzepine, MT7,muscarinic receptor antagonist, and the like with a silicone-typeadhesive or alternatively an acrylate-vinyl acetate adhesive in asolvent exemplified by methylene chloride, ethyl acetate, isopropylmyristate, and propylene glycol. The mixture would then be extruded ontoa polyester-backing film to a uniform thickness of about 100 microns orgreater with a precision wet-film applicator. The solvent is allowed toevaporate in a drying oven and the resulting “patch” is trimmed to theappropriate size.

The pharmaceutical for topical administration or alternatively fortransdermal administration of an agent as described above (e.g.,oxybutynin, pirenzepine, MT7, muscarinic receptor antagonist, and thelike) may additionally incorporate a penetration enhancer and/or athickening agent or gelling agent and/or an emollient and/or anantioxidant and/or an antimicrobial preservative and/or an emulsifyingagent and/or a water miscible solvent and/or an alcohol and/or water.

According to one aspect, the pharmaceutical composition for topicaladministration or transdermal administration of an agent as describedabove (e.g., oxybutynin, pirenzepine, MT7, muscarinic receptorantagonist, and the like) may comprise one or more penetration enhancingagent or co-solvent for transdermal or topical delivery. A penetrationenhancer is an excipient that aids in the diffusion of the activethrough the stratum corneum. Many penetration enhancers also function asco-solvents which are thought to increase the thermodynamic activity orsolubility of the oxybutynin, pirenzepine, MT7, muscarinic receptorantagonist, and the like in the composition. Penetration enhancers arealso known as accelerants, adjuvants or sorption promoters. A suitablepenetration enhancer for use in the pharmaceutical compositions andmethods described herein should: (i) be highly potent, with a specificmechanism of action; (ii) exhibit a rapid onset upon administration;(iii) have a predictable duration of action; (iv) have onlynon-permanent or reversible effects on the skin; (v) be chemicallystable; (vi) have no or minimal pharmacological effects; (vii) bephysically and chemically compatible with other composition components;(viii) be odorless; (ix) be colorless; (x) be hypoallergenic; (xi) benon-irritating; (xii) be non-phototoxic; (xiii) be non-comedogenic;(xiv) have a solubility parameter approximating that of the skin (10.5cal/cm3); (xv) be readily available; (xvi) be inexpensive; and (xvii) beable to formulated in pharmaceutical compositions for topical ortransdermal delivery of an active pharmaceutical agent.

Several classes of chemical compounds, with various mechanisms ofaction, can be used as penetration enhancers. Set forth below arenon-limiting examples of penetration enhancing agents, many of which arealso suitable co-solvents. Sulfoxides, such as dimethylsulfoxide anddecylmethylsulfoxide can be used as penetration enhancing agents.Dimethylsulfoxide enhances penetration in part by increasing lipidfluidity and promoting drug partitioning. In contrast,decylmethylsulfoxide enhances penetration by reacting with proteins inthe skin that change the conformation of the proteins, which results inthe creation of aqueous channels.

Another class of penetration enhancers are alkanones, such as N-heptane,N-octane, N-nonane, N-decane, N-undecane, N-dodecane, N-tridecane,N-tetradecane and N-hexadecane. Alkanones are thought to enhance thepenetration of an active agent by altering the stratum corneum. Afurther class of penetration enhancers are alkanol alcohols, such asethanol, propanol, butanol, 2-butanol, pentanol, 2-pentanol, hexanol,octanol. nonanol, decanol and benzyl alcohol. Low molecular weightalkanol alcohols, i.e., those with 6 or less carbons, may enhancepenetration in part by acting as solubilizing agents, while morehydrophobic alcohols may increase diffusion by extracting lipids fromthe stratum corneum. A further class of penetration enhancers are fattyalcohols, such as oleyl alcohol, caprylic alcohol, decyl alcohol, laurylalcohol, 2-lauryl alcohol, myristyl alcohol, cetyl alcohol, stearylalcohol, oleyl alcohol, linoleyl alcohol and linolenyl alcohol. Polyols,including propylene glycol, polyethylene glycol, ethylene glycol,diethylene glycol, triethylene glycol, dipropylene glycol, glycerol,propanediol, butanediol, pentanediol, hexanetriol, propylene glycolmonolaurate and diethylene glycol monomethyl ether (transcutol), canalso enhance penetration. Some polyols, such as propylene glycol, mayfunction as a penetration enhancer by solvating alpha-kertin andoccupying hydrogen bonding sites, thereby reducing the amount ofactive-tissue binding.

Another class of penetration enhancers are amides, including urea,dimethylacetamide, diethyltoluamide, dimethylformamide,dimethyloctamide, dimethyldecamide and biodegradable cyclic urea (e.g.,1-alkyl-4-imidazolin-2-one). Amides have various mechanisms of enhancingpenetration. For example, some amides, such as urea increase thehydration of the stratum corneum, act as a keratolytic and createhydrophilic diffusion channels. In contrast, other amides, such asdimethylacetamide and dimethylformamide, increase the partition tokeratin at low concentrations, while increasing lipid fluidity anddisrupting lipid packaging at higher concentrations. Another class ofpenetration enhancing agents are pyrrolidone derivatives, such as1-methyl-2-pyrrolidone, 2-pyrrolidone, 1-lauryl-2-pyrrolidone,1-methyl-4-carboxy-2-pyrrolidone, 1-hexyl-4-carboxy-2-pyrrolidone,1-lauryl-4-carboxy-2-pyrrolidone,1-methyl-4-methoxycarbonyl-2-pyrrolidone,1-hexyl-4-methoxycarbonyl-2-pyrrolidone,1-lauryl-4-methoxycarbonyl-2-pyrrolidone, N-methyl-pyrrolidone,N-cyclohexylpyrrolidone, N-dimethylaminopropyl-pyrrolidone,N-cocoalkypyrrolidone and N-tallowalkypyrrolidone, as well asbiodegradable pyrrolidone derivatives, including fatty acid esters ofN-(2-hydroxyethyl)-2-pyrrolidone. In part, pyrrolidone derivativesenhance penetration through interactions with the keratin in the stratumcorneum and lipids in the skin structure. An additional class ofpenetration enhancers are cyclic amides, including1-dodecylazacycloheptane-2-one also known as AZONE© (AZONE is aregistered trademark of Echo Therapuetics Inc., Philadelphia, Pa., USA),1-geranylazacycloheptan-2-one, 1-famesylazacycloheptan-2-one,1-geranylgeranylazacycloheptan-2-one,1-(3,7-dimethyloctyl)-azacycloheptan-2-one,1-(3,7,11-trimefhyldodecyl)azacyclohaptan-2-one,1-geranylazacyclohexane-2-one, 1-geranylazacyclopentan-2,5-dione and1-famesylazacyclopentan-2-one. Cyclic amides, such as AZONE®, enhancethe penetration of active agents in part by affecting the stratumcomeum's lipid structure, increasing partitioning and increasingmembrane fluidity.

Additional classes of penetration enhancers include diethanolamine,triethanolamine and hexamethylenlauramide and its derivatives.

Additional penetration enhancers include linear fatty acids, such asoctanoic acid, linoleic acid, valeric acid, heptanoic acid, pelagonicacid, caproic acid, capric acid, lauric acid, myristric acid, stearicacid, oleic acid and caprylic acid. Linear fatty acids enhancepenetration in part via selective perturbation of the intercellularlipid bilayers. In addition, some linear fatty acids, such as oleicacid, enhance penetration by decreasing the phase transitiontemperatures of the lipid, thereby increasing motional freedom orfluidity of the lipids. Branched fatty acids, including isovaleric acid,neopentanoic acid, neoheptanoic acid, nonanoic acid, trimethyl hexaonicacid, neodecanoic acid and isostearic acid, are a further class ofpenetration enhancers. An additional class of penetration enhancers arealiphatic fatty acid esters, such as ethyl oleate, isopropyl n-butyrate,isopropyl n-hexanoate, isopropyl n-decanoate, isopropyl myristate(“IPM”), isopropyl palmitate and octyldodecyl myristate. Aliphatic fattyacid esters enhance penetration by increasing diffusivity in the stratumcorneum and/or the partition coefficient. In addition, certain aliphaticfatty acid esters, such as IPM, enhance penetration by directly actingon the stratum comeum and permeating into the liposome bilayers therebyincreasing fluidity. Alkyl fatty acid esters, such as ethyl acetate,butyl acetate, methyl acetate, methyl valerate, methyl propionate,diethyl sebacate, ethyl oleate, butyl stearate and methyl laurate, canact as penetration enhancers. Alkyl fatty acid esters enhancepenetration in part by increasing the lipid fluidity.

An additional class of penetration enhancers are anionic surfactants,including sodium laurate, sodium lauryl sulfate and sodium octylsulfate. Anionic surfactants enhance penetration of active agents byaltering the barrier function of the stratum corneum and allowingremoval of water-soluble agents that normally act as plasticizers. Afurther class of penetration enhancers are cationic surfactants, such ascetyltrimethylammonium bromide, tetradecyltrimethylammonium,octyltrimethyl ammonium bromide, benzalkonium chloride,octadecyltrimethylammonium chloride, cetylpyridinium chloride,dodecyltrimethylammonium chloride and hexadecyltrimethylammoniumchloride. Cationic surfactants enhance penetration by adsorbing at, andinteracting with, interfaces of biological membranes, resulting in skindamage. A further class of penetration enhancers are zwitterionicsurfactants, such as hexadecyl trimethyl ammoniopropane sulfonate, oleylbetaine, cocamidopropyl hydroxysultaine and cocamidopropyl betaine.Nonionic surfactants exemplified by Polyxamer 231, Polyxamer 182,Polyxamer 184, Polysorbate 20, Polysorbate 60, BRIJ® 30, BRIJ® 93, BRIJ®96, BRIJ® 99 (BRIJ is a registered trademark of Brij Image & InformationInc., Greensboro, N.C., USA), SPAN® 20, SPAN® 40, SPAN® 60, SPAN® 80,SPAN® 85 (SPAN is a registered trademark of Croda International PLC,East Yorkshire, UK), TWEEN® 20, TWEEN® 40, TWEEN® 60, TWEEN® 80 (TWEENis a registered trademark of Unigema Americas LLC, Wilmington, Del.,USA), Myrj 45, MYRJ® 51, MYRJ® (MYRJ is a registered trademark ofUnigema Americas LLC, Wilmington, Del., USA), and MIGLYOL® 840 (MIGLYOLis a registered trademark of Cremer Oleo GMBH & Co., Hamburg, Fed. Rep.Germany), and the like. Nonionic surfactants enhance penetration in partby emulsifying the sebum and enhancing the thermodynamic activity orsolubility of the active.

Another class of penetration enhancer increase the thermodynamicactivity or solubility of the active, which include, but are not limitedto, n-octanol, sodium oleate, D-limonene, monoolein, cineol, oleyloleate, and isopropryl myristate.

Other penetration enhancers are bile salts, such as sodium cholate,sodium salts of taurocholic acid, glycolic acids and desoxycholic acids.Lecithin also has been found to have penetration enhancingcharacteristics. An additional class of penetration enhancers areterpenes, which include hydrocarbons, such as d-limonene, alpha-pineneand beta-carene; alcohols, such as, alpha-terpineol, terpinen-4-ol andcarvol; ketones, such ascarvone, pulegone, piperitone and menthone;oxides, such as cyclohexene oxide, limonene oxide, alpha-pinene oxide,cyclopentene oxide and 1,8-cineole; and oils such as ylang ylang, anise,chenopodium and eucalyptus. Terpenes enhance penetration in part bydisrupting the intercellular lipid bilayer to increase diffusivity ofthe active and opening polar pathways within and across the stratumcomeum. Organic acids, such as salicylic acid and salicylates (includingtheir methyl, ethyl and propyl glycol derivates), citric acid andsuccinic acid, are penetration enhancers. Another class of penetrationenhancers are cyclodextrins, including 2-hydroxypropyl-beta-cyclodextrinand 2,6-dimethyl-beta-cyclodextrin. Cyclodextrins enhance the permeationof active agents by forming inclusion complexes with lipophilic activesand increasing their solubility in aqueous solutions.

The penetration enhancing agent(s) and/or co-solvent(s) is/are presentin the pharmaceutical composition for topical administration ortransdermal administration of an agent as described above (e.g.,oxybutynin, pirenzepine, MT7, muscarinic receptor antagonist, and thelike) in an amount sufficient to provide the desired level of drugtransport through the stratum comeum and epidermis or to increase thethermodynamic activity or solubility of the oxybutynin, pirenzepine,MT7, muscarinic receptor antagonist, and the like. The one or morepharmaceutically acceptable penetration enhancer and/or co-solvent maybe present in a total amount by weight of about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about5.7%, about 5.8%, about 5.9%, about 6.0%, about 6.1%, about 6.2%, about6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about6.9%, about 7.0%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8.0%, about8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about8.7%, about 8.8%, about 8.9%, about 9.0%, about 9.1%, about 9.2%, about9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about9.9% or about 10%, about 11%, about 12%, about 13%, about 14%, about15%, about 16%, about 17%, about 18%, about 1%, about 20%, about 21%,about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%,about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%,about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%,about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%,about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, or about 95%.

The selected penetration enhancer should be pharmacologically inert,non-toxic, and non-allergenic, have rapid and reversible onset ofaction, and be compatible with the compositions of the invention.Examples of penetration enhancers exemplified by transcutol P, ethylalcohol, isopropyl alcohol, lauryl alcohol, salicylic acid,octolyphenylpolyethylene glycol, polyethylene glycol 400, propyleneglycol, N-decylmethylsulfoxide, DMSO and azacyclo compounds.

In one exemplary embodiment, the present disclosure pertains tocompositions for local administration of a muscarinic acetylcholinereceptor antagonist(s) in a pharmaceutically sufficient amount to treatperipheral neuropathy. As used herein, the term “local” refers to thelimited area near the site of administration, generally the nerves at ornear skin including the epidermis, the dermis, the dermatomes and thelike, with no or limited systemic penetration beyond the skin.

Preferably, the topical delivery is designed to maximize drug deliverythrough the stratum corneum and into the epidermis or dermis ordermatome, and to minimize absorption into the circulatory system. Morepreferable are agents that may be used in topical formulations toprevent the passage of active ingredients or excipients into the lowerskin layers. These so-called skin retardants have been readily developedfor many over-the-counter (OTC) skin formulations, such as sunscreensand pesticides, where the site of action is restricted to the skinsurface or upper skin layers. Research in the area of permeationenhancement or retardation is yielding valuable insights into thestructure-activity relationships of enhancers as well as retardants(Asbill et al., 2000, Percutaneous penetration enhancers: local versustransdermal activity. Pharm. Sci. Tech. Today, 3(1):36-41; Kaushik, etal., 2008, Percutaneous permeation modifiers: enhancement versusretardation. Exp. Opin. Drug Del. 5(5):517-529; Trommer et al., 2006,Overcoming the Stratum Corneum: The Modulation of Skin Penetration. SkinPharmacol. Physiol. 19:106-121) including such compounds as ketorolacstearate, Aminocaprolactam Analogues, Dicarboxylic acid ester, sodiumcitrate, and the like.

The compositions described herein can further comprise componentsusually admixed in such preparations. For example, the compositions mayalso include additional ingredients such as other carriers,moisturizers, oils, fats, waxes, surfactants, thickening agents,antioxidants, viscosity stabilizers, chelating agents, buffers,preservatives, perfumes, dyestuffs, lower alkanols, humectants,emollients, dispersants, sunscreens such as radiation blocking compoundsor particularly UV-blockers, antibacterials, antifungals, disinfectants,vitamins, antibiotics, or other anti-acne agents, as well as othersuitable materials that do not have a significant adverse effect on theactivity of the topical composition. Additional ingredients forinclusion in the carrier are sodium acid phosphate moisturizer, witchhazel extract carrier, glycerin humectant, apricot kernel oil emollient,corn oil dispersant, and the like which are further detailed below.Those of skill in the art will readily recognize additional ingredients,which can be admixed in the compositions described herein.

According to another aspect, the pharmaceutical composition for topicaladministration or for transdermal application of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like maycomprise a thickening or gelling agent suitable for use in thecompositions and methods described herein to increase the viscosity ofthe composition. Suitable agents (also known as gelling agents) areexemplified neutralized anionic polymers or neutralized carbomers, suchas polyacrylic acid, carboxypolymethylene, carboxymethyl cellulose andthe like, including derivatives of Ultrez 10, CARBOPOL® polymers, suchas CARBOPOL® 940, CARBOPOL® 941, CARBOPOL® 954, CARBOPOL® 980, CARBOPOL®981, CARBOPOL® ETD 2001, CARBOPOL® EZ-2 and CARBOPOL® EZ-3. (CARBOPOL isa registered trademark of Lubrizol Advanced Materials Inc., Cleveland,Ohio, USA). As used herein, a “neutralized carbomer” is a synthetic,high molecular weight polymer, composed primarily of a neutralizedpolyacrylic acid. Further, when a base is added to neutralize a carbomersolution, the viscosity of the solution increases. Also suitable areother known polymeric thickening agents, such as PEMULEN® polymericemulsifiers, NOVEON® polycarbophils (PEMULEN and NOVEON are registeredtrademarks of Lubrizol Advanced Materials Inc.), and KLUCEL® (KLUCEL isa registered trademark of Hercules Inc., Wilmington, Del., USA).Additional thickening agents, enhancers and adjuvants may generally befound in Remington's The Science and Practice of Pharmacy as well as inthe Handbook of Pharmaceutical Excipients (Arthur H. Kibbe ed. 2000).Alternatively, the pharmaceutical composition for topical administrationor for transdermal application of oxybutynin, pirenzepine, MT7,muscarinic receptor antagonist, and the like may comprise an anionicpolymer thickening agent precursor, such as a carbomer, which has beencombined with a neutralizer in an amount sufficient to form a gel orgel-like composition with a viscosity greater than 1000 cps as measuredby a Brookfield RV DVII+ Viscometer with spindle CPE-52, torque greaterthan 10% and the temperature maintained at 25° C. Alternatively, theanionic polymer thickening agent precursor may be combined with aneutralizer selected from the group consisting of: sodium hydroxide,ammonium hydroxide, potassium hydroxide, arginine, aminomethy] propanol,tetrahydroxypropyl ethylenediamine, triethanolamine (“TEA”),tromethamine, PEG-15 cocamine, diisopropanolamine, andtriisopropanolamine, or combinations thereof in an amount sufficient toneutralize the anionic polymer thickening agent precursor to form a gelor gel-like composition in the course of forming the composition. Thethickening agents or gelling agents are present in an amount sufficientto provide the desired rheological properties of the composition, whichinclude having a sufficient viscosity for forming a gel or gel-likecomposition that can be applied to the skin of a mammal. The thickeningagent or gelling agent is present in a total amount by weight of about0.1%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.25%, about1.5%, about 1.75%, about 2.0%, about 2.25%, about 2.5%, about 2.75%,about 3.0%, about 3.25%, about 3.5%, about 3.75%, about 4.0%, about4.25%, about 4.5%, about 4.75%, about 5.0%, about 5.25%, about 5.5%,about 5.75%, about 6.0%, about 6.25%, about 6.5%, about 6.75%, about7.0%, about 7.25%, about 7.5%, about 7.75%, about 8.0%, about 8.25%,about 8.5%, about 8.75%), about 9.0%, about 9.25%, about 9.5%, about9.75%, about 10%, about 11%, about 11.5%, about 12%, about 12.5%, about13%, about 13.5%, about 14%, about 14.5% or about 15%, and therebetween.

According to another aspect, the pharmaceutical composition for topicaladministration or for transdermal application of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like maycomprise an emollient. Suitable emollients are exemplified by mineraloil, mixtures of mineral oil and lanolin alcohols, cetyl alcohol,cetostearyl alcohol, petrolatum, petrolatum and lanolin alcohols, cetylesters wax, cholesterol, glycerin, glyceryl monostearate, isopropylmyristate, isopropyl palmitate, lecithin, allyl caproate, altheaofficinalis extract, arachidyl alcohol, argobase EUC, butylene glycol,dicaprylate/dicaprate, acacia, allantoin, carrageenan, cetyldimethicone, cyclome hicone, diethyl succinate, dihydroabietyl behenate,dioctyl adipate, ethyl laurate, ethyl palmitate, ethyl stearate, isoamyllaurate, octanoate, PEG-75, lanolin, sorbitan laurate, walnut oil, wheatgerm oil, super refined almond, super refined sesame, super refinedsoyabean, octyl palmitate, caprylic/capric triglyceride and glycerylcocoate. An emollient, if present, is present in the compositionsdescribed herein in an amount by weight of the composition of about 1%to about 30%, about 3% to about 25%, or about 5% to about 15%.Illustratively, one or more emollients are present in a total amount ofabout 1% by weight, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 1%, about 16%, about 17%, about 18%, about 19%,about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about26%, about 27%, about 28%, about 29%, or about 30%, and therebetween.

According to another aspect, the pharmaceutical composition for topicaladministration or for transdermal application of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like maycomprise an antioxidant. Suitable antioxidants are exemplified by citricacid, butylated hydroxytoluene (BHT), ascorbic acid, glutathione,retinol, a-tocopherol, β-carotene, a-carotene, ubiquinone, butylatedhydroxyanisole, ethyl enediaminetetraacetic acid, selenium, zinc,lignan, uric acid, lipoic acid, and N-acetylcysteine. An antioxidant, ifpresent, is present in the compositions described herein in a totalamount selected from the range of about 0.025% to about 1.0% by weight.

According to another aspect, the pharmaceutical composition for topicaladministration or for transdermal application of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like maycomprise an antimicrobial preservative. Illustrative anti-microbialpreservatives include acids, including but not limited to, benzoic acid,phenolic acid, sorbic acids, alcohols, benzethonium chloride, bronopol,butylparaben, cetrimide, chlorhexidine, chlorobutanol, chlorocresol,cresol, ethylparaben, imidurea, methylparaben, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, potassium sorbate, propylparaben, sodiumpropionate or thimerosal. The anti-microbial preservative, if present,is present in an amount by weight of the composition of about 0.1% toabout 5%, about 0.2% to about 3%, or about 0.3% to about 2%, for exampleabout 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%, about 1.2%,about 1.4%, about 1.6%, about 1.8%, about 2%, about 2.2%, about 2.4%,about 2.6%, about 2.8%, about 3.0%, about 3.2%, about 3.4%, about 3.6%,about 3.8%, about 4%, about 4.2%, about 4.4%, about 4.6%, about 4.8%, orabout 5%.

According to another aspect, the pharmaceutical composition for topicaladministration or for transdermal application of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like maycomprise one or more emulsifying agents. The term “emulsifying agent”refers to an agent capable of lowering surface tension between anon-polar and polar phase and includes self emulsifying agents. Suitableemulsifying agents can come from any class of pharmaceuticallyacceptable emulsifying agents exemplified by carbohydrates, proteins,high molecular weight alcohols, wetting agents, waxes and finely dividedsolids. The optional emulsifying agent, if present, is present in acomposition in a total amount of about 1% to about 25%, about 1% toabout 20%, or about 1% to about 15% by weight of the composition.Illustratively, one or more emulsifying agents are present in a totalamount by weight of about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about25%.

According to another aspect, the pharmaceutical composition for topicaladministration or for transdermal application of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like maycomprise a water miscible solvent exemplified by propylene glycol. Asuitable water miscible solvent refers to any solvent that is acceptablefor use in a pharmaceutical composition and is miscible with water. Ifpresent, the water miscible solvent is present in a composition in atotal amount of about 1% to about 95%, about 2% to about 75%, about 3%to about 50%, about 4% to about 40%, or about 5% to about 25% by weightof the composition.

According to another aspect, the pharmaceutical composition for topicaladministration or for transdermal application of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like maycomprise one or more alcohols. In a further embodiment, the alcohol is alower alcohol. As used herein, the term “lower alcohol,” alone or incombination, means a straight-chain or branched-chain alcohol moietycontaining one to about six carbon atoms. In one embodiment, the loweralcohol contains one to about four carbon atoms, and in anotherembodiment the lower alcohol contains two or three carbon atoms.Examples of such alcohol moieties include methanol, ethanol, ethanol USP(i.e., 95% v/v), n-propanol, isopropanol, n-butanol, isobutanol,sec-butanol, and tert-butanol. As used herein, the term “ethanol” refersto C2H5OH. It may be used as dehydrated alcohol USP, alcohol USP or inany common form including in combination with various amounts of water.If present, the alcohol is present in an amount sufficient to form acomposition which is suitable for contact with a mammal. For example, ina total amount by weight of about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%,about 25%.

According to another aspect, the pharmaceutical composition for topicaladministration or for transdermal application of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like maycomprise water separately in a quantity or amount sufficient to achievethe desired weight of the pharmaceutical composition.

Oxybutynin, pirenzepine, MT7, muscarinic receptor antagonist, and thelike comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4%, about4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about5.9%, about 6%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7%, about7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about7.7%, about 7.8%, about 7.9%, about 8%, about 8.1%, about 8.2%, about8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about8.9%, about 9%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%,about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%,about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90% or about 95% by weight of the pharmaceuticalcomposition for topical application or for transdermal application.

Another embodiment pertains to pharmaceutical compositions comprisingoxybutynin, pirenzepine, MT7, muscarinic receptor antagonist, and thelike formulated for parenteral administration by injection. Theinjectable pharmaceutical compositions of the present disclosurecomprise a suitable carrier solution exemplified by sterile water,saline, and buffered solutions at physiological pH. Suitable bufferedsolutions are exemplified by Ringer's dextrose solution and Ringer'slactated solutions. The carrier solution may comprise in a total amountby weight of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%,about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%,about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%,about 1.8%, about 1.9%, about 2.0%>, about 2.1%, about 2.2%, about 2.3%,about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%,about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%,about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%,about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%,about 4.8%, about 4.9%, about 5.0%, about 5.1%, about 5.2%, about 5.3%,about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%,about 6.0%, about 6.1%, about 6.2%, about 6.3%>, about 6.4%, about 6.5%,about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7.0%, about 7.1%,about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%,about 7.8%, about 7.9%, about 8.0%, about 8.1%, about 8.2%, about 8.3%,about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%,about 9.0%, about 9.1%, about 9.2%), about 9.3%, about 9.4%, about 9.5%,about 9.6%, about 9.7%, about 9.8%, about 9.9% or about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%,about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%,about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%,about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%,about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%,about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%.

According to one aspect, the injectable pharmaceutical compositions mayadditionally incorporate one or more non-aqueous solvents exemplified bypropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters exemplified by ethyl oleate.

According to another aspect, the injectable pharmaceutical compositionsmay additionally incorporate one or more of antimicrobials,anti-oxidants, chelating agents and the like.

The injectable pharmaceutical compositions may be presented in unit-doseor multi-dose containers exemplified by sealed ampules and vials. Theinjectable pharmaceutical compositions may be stored in a freeze-dried(lyophilized) condition requiring the addition of a sterile liquidcarrier, e.g., sterile saline solution for injections, immediately priorto use.

Another embodiment pertains to pharmaceutical compositions comprisingoxybutynin, pirenzepine, MT7, muscarinic receptor antagonist, and thelike formulated for oral administration. The oral pharmaceuticalcompositions may be provided as capsules or tablets; as powders orgranules; as solutions, syrups or suspensions (in aqueous or non-aqueousliquids). Tablets or hard gelatine capsules may comprise, for example,lactose, starch or derivatives thereof, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate, stearic acid or saltsthereof. Soft gelatine capsules may comprise, for example, vegetableoils, waxes, fats, semisolid, or liquid polyols, etc. Solutions andsyrups may comprise, for example, water, polyols and sugars. Theoxybutynin, pirenzepine, MT7, muscarinic receptor antagonist, and thelike may be coated with or admixed with a material (e.g., glycerylmonostearate or glyceryl distearate) that delays disintegration oraffects absorption of the active agent in the gastrointestinal tract.Thus, for example, the sustained release of an active agent may beachieved over many hours and, if necessary, the active agent can beprotected from being degraded within the gastrointestinal tract. Takingadvantage of the various pH and enzymatic conditions along thegastrointestinal tract, pharmaceutical compositions for oraladministration may be formulated to facilitate release of an activeagent at a particular gastrointestinal location.

The pharmaceutical compositions described herein are used in a“pharmacologically effective amount.” A “pharmacologically effectiveamount” is the amount of oxybutynin, pirenzepine, MT7, muscarinicreceptor antagonist, and the like in the composition which is sufficientto deliver a therapeutic amount of the active agent during the dosinginterval in which the pharmaceutical composition is administered.Accordingly, the amount of the pharmaceutical composition administeredto deliver a therapeutically effective amount of oxybutynin,pirenzepine, MT7, muscarinic receptor antagonist, and the like is about0.01 g, about 0.05 g, about 0.1 g, about 0.2 g, about 0.3 g, about 0.4g, about 0.5 g, about 0.6 g, about 0.7 g, about 0.8 g, about 0.9 g,about 1 g, about 1.1 g, about 1.2 g, about 1.3 g, about 1.4 g, about 1.5g, about 1.6 g, about 1.7 g, about 1.8 g, about 1.9 g, about 2 g, about2.1 g, about 2.2 g, about 2.3 g, about 2.4 g, about 2.5 g, about 2.6 g,about 2.7 g, about 2.8 g, about 2.9 g, about 3 g, about 3.1 g, about 3.2g, about 3.3 g, about 3.4 g, about 3.5 g, about 3.6 g, about 3.7 g,about 3.8 g, about 3.9 g, about 4 g, about 4.1 g, about 4.2 g, about 4.3g, about 4.4 g, about 4.5 g, about 4.6 g, about 4.7 g, about 4.8 g,about 4.9 g, about 5 g, about 5.1 g, about 5.2 g, about 5.3 g, about 5.4g, about 5.5 g, about 5.6 g, about 5.7 g, about 5.8 g, about 5.9 g,about 6 g, about 6.1 g, about 6.2 g, about 6.3 g, about 6.4 g, about 6.5g, about 6.6 g, about 6.7 g, about 6.8 g, about 6.9 g, about 7 g, about7.1 g, about 7.2 g, about 7.3 g, about 7.4 g, about 7.5 g, about 7.6 g,about 7.7 g, about 7.8 g, about 7.9 g, about 8 g, about 8.1 g, about 8.2g, about 8.3 g, about 8.4 g, about 8.5 g, about 8.6 g, about 8.7 g,about 8.8 g, about 8.9 g, about 9 g, about 9.1 g, about 9.2 g, about 9.3g, about 9.4 g, about 9.5 g, about 9.6 g, about 9.7 g, about 9.8 g,about 9.9 g or about 10 g.

The following examples are provided to more fully describe thedisclosure and are presented for non-limiting illustrative purposes.

EXAMPLES

The fundamental hypothesis is that blocking endogenous cholinergicconstraint using a muscarinic receptor antagonist will promote re-growthof neurons in patients with established peripheral neuropathy. Thisoccurs via activation of AMPK and PGC-la and the subsequent augmentationof mitochondrial function. This pathway comprises a proximal stepinvolving M1 receptor antagonist-dependent activation of CaMKKO (orCaMKK2). This hypothesis is supported by extensive preclinical datausing in vitro and in vivo model systems. Experiments show thatmuscarinic receptor antagonists dose-dependently promote neuriteoutgrowth from sensory neurons derived from adult rodents (FIG. 1). Aspecific requirement for M1 receptor antagonism is confirmed by theincreased neurite outgrowth from sensory neurons derived from M1R KOmice.

Example 1

In previous work, we showed that pirenzepine can prevent and reversesmall fiber neuropathy in diabetes. Now we demonstrate that pirenzepinecan also prevent large fiber motor nerve conduction velocity (MNVC)slowing (FIG. 2(A)) and sensory nerve conduction velocity (SNCV) slowing(FIG. 2(B)) in streptozotocin-induced (STZ-induced) diabetic rats. TheSTZ rat is a model of type 1 diabetes. Rats were treated with 5 mg/kg(s.c.) pirenzepine for 8 weeks following the method taught by Calcutt etal. (2003, Therapeutic efficacy of sonic hedgehog protein inexperimental diabetic neuropathy. J. Clin. Invest. 111:507-514). Nerveconduction velocities (NCV) were determined following the methods taughtby Mizisin et al. (2004, Ciliary Neurotrophic Factor Improves NerveConduction and Ameliorates Regeneration Deficits in Diabetic Rats.Diabetes 53:1807-12). The data show that diabetes caused time-dependentslowing of large fiber motor and sensory NCV (both p<0.01 vs control at8 weeks of diabetes) and these disorders were attenuated by pirenzepinetreatment (both p<0.05 vs vehicle-treated diabetic rats). All data aregroup mean±SEM (N=5-6 group). Statistical analysis by one-way ANOVA withDunnett's post-hoc test. These date demonstrate that pirenzepine is ableto protect function of large-diameter myelinated fibers of both motorand sensory nerves.

Example 2

Subsets of cultured adult sensory neurons derived from age-matchedcontrol rats or 3-5 month STZ-induced diabetic rats were treated with a1.0 μM pirenzepine-HCl dosage. The first subset was processedimmediately while the second, third and fourth subsets of the neuronsfrom the control rats and the diabetic rats were processed after 15 min,after 30 min, and after 60 min respectively following treatment withpirenzepine. The cell cultures were lysed and then used to prepareWestern blots which were then probed with antibodies againstphosphorylated AMPK (P-AMPK), total AMPK (T-AMPK) and total ERK (T-ERK).FIG. 3(A) shows the gels produced from the cultures from age-matchedcontrol rats while FIG. 3(C) shows the gels produced from the culturesfrom diabetic rats. The data shown in in FIGS. 3(B) and 3(D), where theY-axis represents levels of protein quantified from the blots in (A) and(C) and normalized to control, show that pirenzepine significantlyactivated P-AMPK in cultures from normal rats (FIG. 3(B)) and in type 1diabetic rats (FIG. 3(D)). Values are means±SEM, N=3/group. Statisticalsignificance was analyzed using one-way ANOVA with Tukey's post-hoctest.

Example 3

Adult sensory neuron cultures derived from STZ-induced diabetic ratswere transduced for two days after treatment with 1.0 μM pirenzepine,with two adenovirus over-expressing dominant negative mutants of AMPK,i.e., AMPK subunit α1 and α2 mutants named “AdDN1” and “AdDN2” (theadenovirus over-expressing dominant negative mutants were gifts from Dr.Jason Dyck, University of Alberta) following the procedure taught by RoyChowdhury et al., (2012). FIGS. 4(A) and 4(B) are micrographs of GFPfluorescence from adult rat sensory neuron cultures transduced with thecontrol adenoviral vector expressing only GFP wherein FIG. 4(A) did notreceive a pirenzepine treatment whereas FIG. 4(B) did. FIGS. 4(C) and4(D) are micrographs of GFP fluorescence from adult rat sensory neuroncultures transduced with the “AdDN” dominant negative AMPK mutantwherein FIG. 4(C) did not receive a pirenzepine treatment whereas FIG.4(D) did. The data in FIGS. 5(A) and 5(B) show that blockage of AMPKsignaling using adenovirus-delivered dominant negative mutantscompletely inhibited pirenzepine-induced neurite outgrowth.

Example 4

PGC-la transcriptional activity was determined in dissociated adultsensory neurons derived from STZ-induced diabetic rats by transfectionfor two days with luciferase-based reporter plasmids by following themethods taught in the AMAXA® NUCLEOFECTOR® II Manual (www.amaxa.com;AMAX and NUCLEOFECTOR are registered trademarks of Amaxa GmBH Corp.,Koln, Fed. Rep. Germany) and by Saleh et al. (2013, Ciliary neurotrophicfactor activates NF-κB to enhance mitochondrial bioenergetics andprevent neuropathy in sensory neurons of streptozotocin-induced diabeticrodents. Neuropharmacology 65: 65-75) (the luciferase-based reporterplasmids were a gift from Dr. Michael Czubryt, University of Manitoba).FIG. 6 shows that 1 μM pirenzepine enhanced transcriptional activity ofPGC-la in cultured adult sensory neurons derived from STZ-induceddiabetic rats (means±SEM of N=3 replicate cultures. Oneway ANOVA withTukey's postdoc test. Values normalized to control reporter (PGL3)expression). However, mutant PGC-1α plasmids exhibited zero activity inthis assay. Accordingly, these data demonstrate that pirenzepine signalsvia activation of the AMPK/PGC-la axis to drive neurite outgrowth.

Example 5

Mitochondrial respiration in adult mouse sensory neuron cultures fromwild type control mice, M1 receptor knockout mice (MIR KO), and fromdiabetic mice that received dosing with 1 μM of VU0255035 for 3 h, wereassessed following the methods taught by Roy Chowdhury et al. (2012).The MIR knockout mice were a gift from Dr. Jurgen Wess (MolecularSignaling Section, Laboratory of Bioorganic Chemistry, NationalInstitute of Diabetes and Digestive and Kidney Diseases, NationalInstitutes of Health).

The data in FIG. 7(A) show that the oxygen consumption rate (OCR) wassignificantly augmented in neuron cultures derived from M1 receptorknockout mice (MIR KO) while the data in FIG. 7(B) show that treatmentwith VU0255035 resulted in increased OCR in neuron cultures derived fromSTZ-induced diabetic rats. The data in FIGS. 8(A), 8(B) indicate thatthe coupling efficiencies in the neuron cultures from wild type mice,from MIR KO mice, and from diabetic mice that received dosing withVU0255035 were not dissimilar as also were their respiratory controlrations (FIGS. 8(C), 8(D)). However, the spare respirator capacity wassignificantly increased in neuron cultures from the MIR KO mice and fromthe VU0255035-treated cultures from diabetic rats compared to controls(FIGS. 8(E), 8(F)) (the data shown in FIGS. 8(A)-8(F) are means±SEM ofN=4-5 replicate cultures. Groups were compared using Student's t-test).This bioenergetics parameter, which is depressed in diabetes, is relatedto the capacity of cells to conduct respiration under conditions ofstress or high ATP demand. In this regard we propose the stress is thepresence of high intracellular glucose concentrations. Therefore, wepropose that muscarinic receptor antagonism enhances AMPK/PGC-lasignaling to augment mitochondrial function that provides the energy inthe form of ATP (from oxidative phosphorylation) for induction of axonregeneration.

Example 6

The highly specific M1 receptor antagonist, MT7 (muscarinic toxin 7),enhanced neurite outgrowth in cultured adult rat sensory neurons (FIGS.9(A), 9(B), 10). The data in FIG. 10 are means±SEM of 9 replicatecultures. Significant differences were determined by one-way ANOVA withDunnett's post-hoc test.

The data shown in FIGS. 11(A), 11(B) demonstrate that MT7 treatmentactivated transcription of PGC-1α in cultured adult sensory neuronsderived from STZ-induced diabetic rats, while co-treatment with 0.3 M ofCompound C (CC), a pharmacological inhibitor of AMPK, blocked thestimulatory effects of MT7 (the data points in FIG. 11 are means±SEM ofN=3 replicate cultures. Significant differences were determined byoneway ANOVA with Tukey's post-hoc test). These data confirm that theAMPK/PGC-1α pathway is modulated by the M1 receptor, with specificblockade of the M1 receptor causing enhanced activity of AMPK/PGC-la.

The data in FIG. 12 show that pirenzepine-induced neurite outgrowth incultured adult sensory neurons derived from rats was dose-dependentlyinhibited by the CaMKK inhibitor, STO-609 (data are means±SEM of N=8-10replicate cultures. Significant differences were determined by onewayANOVA followed by Dunnett's post-hoc test).

The data in FIGS. 13(A)-13(D) show that blockade of CaMKK using 1.0 MSTO-609 inhibited the pirenzepine-induced or MT7-induced enhancement ofAMPK phosphorylation in cultured adult sensory neurons derived fromage-matched control rats (data are means±SEM of N=3 replicate cultures.Significant differences were determined by oneway ANOVA followed byDunnett's post-hoc test).

These data confirm that the induction of CaMKK, specifically CaMKKO (orCaMKK2) is a proximal step in the pathway leading from blockade of theM1 receptor to activation of AMPK.

Example 7

The data in FIG. 14 show that oxybutynin, a broad spectrum M1, M2 and M3receptor antagonist, elevated neurite outgrowth in cultured adultsensory neurons. This confirms that less selective antagonists ofmuscarinic receptors are also efficacious in enhancing axon regenerationin vitro (the data are means±SEM of N=6 replicate cultures. Significantdifferences were determined with a oneway ANOVA followed by Dunnett'spost-hoc test).

The muscarinic receptor antagonist oxybutynin was tested for efficacyagainst a functional measure of sensory neuropathy in the db/db mousemodel of type 2 diabetes (FIG. 15). Thermal latency was analyzed astaught by Calcutt et al. (2004, Prevention of sensory disorders indiabetic Sprague-Dawley rats by aldose reductase inhibition or treatmentwith ciliary neurotrophic factor. Diabetologia 47:718-24). Female db/dbmice were allowed to develop diabetes and small fiber sensory neuropathywas indicated by paw thermal hypoalgesia when compared to age-matchednon-diabetic C57 mice (p<0.01). Some diabetic mice were then treatedwith oxybutynin (Oxy: 2% in hydrogel) applied to the right paw for 20min/day 5 days a week, while others were treated with hydrogel vehiclealone. Treatment with oxybutynin for 8 weeks reversed paw thermalhypoalgesia in db/db mice so that values were significantly (p<0.01)lower than in vehicle treated mice, although they remained significantly(p<0.05) higher than in the non-diabetic C57 mice. Data are groupmean±SEM of N=8-9/group. At week 8, all other groups=p<0.01 vs db/dbgroup by one-way ANOVA followed by Dunnett's post-hoc test.

These data show that thermal hypoalgesia was reversed in adult type 2diabetic (db/db) mice treated with topical oxybutynin within 4-8 weeksof treatment. Thus this drug, which is currently used by humans to treatover-active bladder syndrome, was effective in reversing small fiberneuropathy in type 2 diabetes.

In this same study, treatment with oxybutynin was also effective inpreventing loss of intraepidermal nerve fibers (IENF; FIG. 16(A)) andcorneal nerves (FIG. 16(B)). IENF levels were quantified as taught byBeiswenger et al. (2008, Epidermal nervefiber quantification in theassessment of diabetic neuropathy. Acta Histochem. 110:351-63). The datashown in FIG. 16(A) are group mean±SEM of N=6-8/group. Significantdifferences were determined with a oneway ANOVA followed by Dunnett'spost-hoc test. Vehicle-treated diabetic (db/db) mice showed significant(p<0.05) reductions in density of IENF in paw skin that was attenuatedby oxybutynin treatment so that values were not significantly differentfrom controls. The data shown in FIG. 16(B) show the effects ofoxybutynin on levels of nerve fibers within the cornea of db/db diabeticmice calculated as the % occupancy of the cornea by nerve fibers. After8 weeks of topical oxybutynin treatment, the eyes were assessed usingcorneal confocal microscopy as taught by Chen et al. (2013, Repeatedmonitoring of corneal nerves by confocal microscopy as an index ofperipheral neuropathy in type-1 diabetic rodents and the effects oftopical insulin. J. Periph. Nerv. Syst.18; 306-315). Vehicle treateddiabetic (db/db) mice showed a significant (p<0.05) reduction in nervefiber occupancy in the cornea and thist was attenuated by oxybutynintreatment so that values were not significantly different from controls.

These data confirm that oxybutynin delivered via a topical route is ableto reverse or prevent an array of small fiber deficits in type 2diabetic mice. In Swiss Webster mice with type 1 diabetes that wasinduced by STZ, the systemic delivery of 3 mg/kg s.c. or 10 mg/kg s.c.oxybutynin for 9 weeks after an initial period of 8 weeks of untreateddiabetes was also able to reverse thermal hypoalgesia (FIG. 17).Collectively, these data support the potential of selective M1Rantagonists, including pirenzepine, VU0255035, MT7 and oxybutynin amongothers, to reverse established small fiber neuropathy in diabetes inaddition to preventing functional indices of large fiber neuropathy(FIG. 2).

Example 8

Given the phenotypic parallels between peripheral neuropathy induced bydiabetes and chemotherapeutic agents, we have demonstrated that reducedneurite outgrowth of sensory neurons when exposed in vitro to paclitaxel(FIG. 18(A)) or to oxaliplatin (FIG. 18(B)) was prevented bypirenzepine. Adult sensory neurons from normal rats were cultured for 24hours in the presence of (A) paclitaxel (PX; 0.1 μM or 0.3 μM), or (B)oxaliplatin (OX; 3.0 μM). From time of plating some cultures weretreated with 0.1, 1.0 or 10 μM pirenzepine (PZ). Levels of total neuriteoutgrowth were determined and presented as means±SEM, N=6-8/group.Significant differences were assessed in (A) by t-test, and in (B) byone-way ANOVA with Dunnett's post-hoc test.

Swiss Webster mice were treated with paclitaxel (taxol; 5 mg/kg on days1, 3, 5 and 7) to induce peripheral neuropathy. Some paclitaxel-treatedmice were also treated with pirenzepine (Pz: 10 mg/kg/day s.c.) for 4weeks following paclitaxel treatment, while others were treated withvehicle alone. FIG. 19 shows paw thermal response latency (left panel)and paw 50% tactile response threshold (right panel) in mice treatedwith paclitaxel±pirenzepine (PZ). Four weeks after paclitaxel treatmentmice developed significant (p<0.01) paw thermal hyperalgesia and tactileallodynia, indicative of painful neuropathy transduced by small andlarge sensory fibers respectively. Data points are group mean ofn=9-12/group±SEM. Significant differences were determined with a onewayANOVA followed by Dunnett's post-hoc test. The data shown in FIG. 19demonstrate that paclitaxel (taxol)-treated mice developed peripheralneuropathy characterized by tactile allodynia and thermal hyperalgesiathat was prevented by daily treatment with pirenzepine dosing at 1mg/kg/day s.c.

Example 9

Another chemotherapeutic agent, dicholoroacetate (DCA), was alsoinvestigated and was shown to induce a peripheral neuropathy in micethat was characterized by sensory loss. Swiss Webster mice were treatedwith DCA (1 mg/kg/day s.c.) for 8 weeks to induce peripheral neuropathyfollowing the method described for rats by Calcutt et al. (2009,Peripheral neuropathy in rats exposed to dichloroacetate. J. Neuropath.Exp. Neurol. 68:985-93). Some DCA-treated mice were also treated withpirenzepine (Pz: 10 mg/kg/day s.c.) for the duration of the study whileothers were treated with vehicle alone. After 8 weeks of DCA treatment,mice developed significant (p<0.01) paw thermal hypoalgesia (FIG. 20(A))indicative of small sensory fiber dysfunction, that was accompanied bysignificant (p<0.01) depletion of paw skin IENF (FIG. 20(B)). Bothdisorders were prevented by treatment with pirenzepine. Pirenzepine alsodelayed onset of MNCV slowing, an index of large fiber dysfunction, inDCA-treated mice after 4, but not 8, weeks of exposure (FIG. 20(C)). Thedata shown in FIGS. 20(A)-20(C) represent group mean±SEM withn=9-12/group. Significant differences were determined by oneway ANOVAfollowed by Dunnett's post-hoc test

Taken together, these data demonstrate that treatment with pirenzepineprevented indices of painful peripheral neuropathy as illustrated bythermal hyperalgesia and tactile allodynia, and also indices ofdegenerative neuropathy as illustrated by thermal hypoalgesia, IENF lossand MNCV slowing. Thus in two different models of chemotherapy-inducedperipheral neuropathy (CIPN), systemic treatment with pirenzepine wasable to protect from small and large fiber dysfunction and/or fiberloss.

Example 10

To extend our study of muscarinic receptor antagonists we treatedSTZ-induced diabetic mice with a broad spectrum drug, atropine. It isknown that atropine blocks the activity of all muscarinic receptors.

Groups of adult Swiss Webster mice were made diabetic with STZ and thentreated 5 days/wk with atropine delivered topically to one eye (2%solution) or topically to one paw (2% gel) for 12 weeks. Measurementswere made on both the treated and untreated hind paw. Indices of largemotor fiber function (MNCV; FIG. 21) and sensory sensory fiber function(paw thermal latency; FIG. 22) were measured and compared to those ofage-matched control mice that were treated in an equivalent manner.Diabetic mice showed significantly (p<0.01) reduced large fiber MNCVcompared to control mice (FIG. 21), whereas diabetic mice treated withatropine delivered either to the eye or the foot had values notdifferent to controls treated in the same manner (FIG. 21). Diabeticmice also showed significant (p<0.01) paw small sensory fiber-mediatedthermal hypoalgesia compared to control mice (FIG. 22), whereas diabeticmice treated with atropine delivered either to the eye or the foot hadvalues not different to controls treated in the same manner (FIG. 22).The data shown in FIGS. 21 and 22 are group means±SEM with N=6-10/group.Significant differences were determined by unpaired t-test to comparecontrol and diabetic animals under each condition (vehicle, atropine tothe eye, atropine to the foot).

These data confirm that delivery of atropine via the eye (by drops) orvia the skin (topical application in hydrogel) caused protection fromlarge and small fiber dysfunction in type 1 diabetic mice. Thereforeselective M1 receptor antagonists, such as pirenzepine, VU0255035 andMT7, as well as broad-spectrum muscarinic receptor antagonists, such asoxybutynin and atropine, are efficacious in preventing and reversingperipheral neuropathy.

Example 11

To further extend our investigation of the actions of muscarinicreceptor antagonists on nerve growth, we treated normal mice with the M1receptor-specific antagonist MT7 by daily delivery to the eyes for 11days and used confocal microscopy to provide a non-invasive andtherefore iterative assessment of corneal nerve density.

A group (n=8) of adult Swiss Webster mice received daily treatment withMT7, which was given by eye drops (20 μl volume) in saline. Nerveoccupancies in the sub-basal nerve plexus (SBNP 1-5) and upper stromal(Stroma 1-10) regions of the cornea were measured on day 2 and day 11 oftreatment (FIG. 23). Nerve occupancy increased significantly (p<0.05 bypaired test) in both regions by day 11 (white bars) when compared tovalues measured on day 2 (black bars). Data are group means (N=8)±SEM.

These data demonstrate the efficacy of MT7 to promote growth of smallsensory neurons of the cornea in vivo.

Example 12

To further extend our investigation of the actions of muscarinicreceptor antagonists on nerve growth, cultured adult rat sensory neuronswere treated for 24 hours with recombinant gp120, an external coatprotein of HIV that causes toxicity to sensory neurons in HIV neuropathy(FIG. 24). This toxicity leads to axon degeneration and neuronal celldeath. Cultures were treated with 1 μM pirenzpeine and total neuriteoutgrowth determined. Pirenzepine treatment afforded complete protectionfrom gp120-induced neurite degeneration (P<0.05 by oneway ANOVA withTukeys post-hoc test). MT7 also exhibited approximately 50% level ofprotection from gp120 treatment (not shown). Values are means±SEM, n=6.

1. A method for treating a chemotherapy-induced peripheral neuropathy ina subject, the method comprising: identifying a subject with achemotherapy-induced peripheral neuropathy; and administering thesubject a composition comprising: an effective amount of a muscarinicacetylcholine receptor M1 antagonist, and a pharmacologically acceptablecarrier and/or an excipient.
 2. The method of claim 1, wherein thecomposition is administered by a subcutaneous injection, orally, and/ortopically.
 3. The method of claim 2, when administered topically, thecomposition additionally comprises one or more of a skin penetrationenhancer, an emollient, an emulsifying agent, a water miscible solvent,an alcohol, and mixtures thereof.
 4. The method of claim 3, wherein theskin penetration enhancer is in a concentration capable of enhancingpenetration of the muscarinic acetylcholine receptor M1 antagonistthrough the skin.
 5. The method of claim 4, wherein the skin penetrationenhancer is a non-ionic skin-penetration enhancer.
 6. The method ofclaim 4, wherein the skin penetration enhancer is about 0.5% by weightof the composition.
 7. The method of claim 1, wherein the composition isone of a lotion, a cream, a gel, and a viscous fluid.
 8. The method ofclaim 7, wherein the composition is administrable from a transdermalpatch.
 9. The method of claim 1, wherein the muscarinic acetylcholinereceptor M1 antagonist is selected from the group consisting ofaprophen, atropine, benactyzine, benztropine, bipreiden, cyclopentolate,dexetimide, dicyclomine, diphenylacetoxy-N-methyl-piperidine methiodide,emepronium, glycopyrrolate, glycopyrronium bromide,hexahydrosiladifenidol, hyoscine, iptratropium bromide, ipratropium,tropicamide, nitrocaramiphen, nuvenzepine, octylonium, orphenadrine,oxyphenonium, pirenzepine, procyclidine, propantheline, quinidine,quinuclidinyl benzilate, rispenzepine, scopolamine, spirotramine,telenzepine, tolterodine, trihexyphenidyl, (−)-5′-ET126,4-diphenylacetoxy-1,1-dimethylpiperidinium,4-fluorhexahydrosiladifenidol, N-desmethylclozapine,0-methoxy-sila-hexocyclium, p-fluorotrihexyphenidyl, R-procyclidine, DAU5750, MB-OXTP, McN-A-343, MDL74019DG, MT7, PD102807, PD150714,VU0255035, salts thereof, and mixtures thereof.
 10. The method of claim1, wherein the muscarinic acetylcholine receptor M1 antagonist isselected from a group consisting of compounds having a chemicalstructure shown in Formula I:

wherein R₁ combines with R₂ to form a 5-membered ring or a 6-memberedring, or alternatively, is one of a 5-membered unsaturated ring or a6-membered unsaturated ring; R₂ combines with R₁ to form a 5-memberedring or a 6-membered ring, or alternatively, is a 5-membered unsaturatedring or a 6-membered unsaturated ring; R₃ is a H-piperidinyl group or a2-piperidinyl group or a 3-piperidinyl or a 4-piperidinyl group, or a2-piperazinyl group or a 3-piperazinyl, linked by a methyl group or anethyl group or a propyl group or a butyl group; R₄ is a hydrogen ion ora chloride ion; R₅ is a hydrogen ion or a chloride ion; R₆ is a hydrogenion or a chloride ion; and R₇ is a hydrogen ion or a chloride ion. 11.The method of claim 1, wherein the muscarinic acetylcholine receptor M1antagonist is selected from a group consisting of compounds having achemical structure shown in Formula II:

wherein X is a methyl group or a primary amine group or a secondaryamine group or a tertiary amine group; R₁ is or

R₂ is a hydroxyl ion or a hydrogen ion or a ketone; and R₃ is a hydroxylion or a hydrogen ion or a ketone.
 12. The method of claim 1, whereinthe muscarinic acetylcholine receptor M1 antagonist comprises: (a)pirenzepine, a pirenzepine salt, or a pirenzepine hydrate; (b)telenzepine salt, a telenzepine derivative, or a combination thereof;(c) an atropine salt, an atropine derivative, or a combination thereof;(d) a VU255035 salt, a VU255035 derivative, or a combination thereof;and/or (e) muscarinic toxin
 7. 13. The method of claim 1, wherein themuscarinic acetylcholine receptor M1 antagonist comprises from 0.1% to10% by weight of the composition.
 14. The method of claim 1, wherein thesubject is also being treated for cancer with a chemotherapeutic agent.15. The method of claim 14, wherein the chemotherapeutic agent isoxaliplatin of Paclitaxel.
 16. The method of claim 15, wherein theacetylcholine receptor M1 antagonist is pirenzepine.